guanosine-triphosphate and 5--guanylylmethylenebisphosphonate

Topics: Adenosine Triphosphate; Animals; Cattle; Cell Line; Dimerization; Fluorescent Antibody Technique, Indirect; Guanosine Triphosphate; Kinesins; Microtubules; Multigene Family; Nerve Tissue Proteins; Protein Binding; Protein Isoforms; Recombinant Fusion Proteins; Spodoptera; Tetrahymena; Tubulin

Topics: Biopolymers; Cell Fractionation; Cell-Free System; Cytosol; Endosomes; Fluorescent Dyes; Guanosine Triphosphate; HeLa Cells; Humans; Intracellular Membranes; Microscopy, Video; Microtubules; Molecular Motor Proteins; Movement; Paclitaxel; Tubulin

Microtubules exhibit dynamic instability, switching between persistent states of growth and shortening at their ends. The switch between growth and shortening has been proposed to depend on end conformation where growing ends have "straight" tubulin protofilaments stabilized by a terminal cap of GTP-tubulin, while-shortening ends have lost their GTP-tubulin cap, allowing terminal GDP-tubulin dimers to curve inside-out and peel rapidly away from the microtubule lattice. This "conformational cap" model predicts that tubulin dissociation from shortening ends is a two-step process where the average lengths of curved GDP-tubulin protofilaments at a depolymerizing end will depend on the ratio of the rate of peeling to the rate of breakage of the longitudinal bonds between adjacent curved dimers. We have tested this model for the plus and minus ends of microtubules assembled with pure porcine tubulin off the ends of axoneme fragments in standard assembly buffer. Individual microtubule ends were imaged using video-enhanced differential interference contrast light microscopy. The rate of rapid shortening was systematically increased by isothermal dilution into assembly buffer containing various concentrations of Mg2+ or Ca2+ ions. At 1 mM Mg2+ and no Ca2+, shortening occurred at 20 (plus) and 45 (minus) microns/min. The ends appeared similar in contrast to growing ends and the core of the microtubule and the ends appeared blunt or slightly frayed by negative stain electron microscopy. Above 20 mM Mg2+ or above 5 mM Ca2+, microtubule shortening occurred at 60 (plus) and 115 (minus) microns/min or faster and "knobs" were distinctly visible at depolymerizing ends, particularly at the faster minus ends, and knob contrast remained constant during many micrometers of rapid shortening. Negative stain electron microscopy revealed that these knobs were "blossoms" of inside-out curved protofilaments, some extending for several helical turns (30 to 60 dimers in length) at constant curvature from the ends. At these high shortening velocities, the peeling of curved protofilaments was confined to within several dimers of the end of the microtubule cylinder, suggesting that dimer curling and protofilament peeling is constrained to the tip by interactions between adjacent straight protofilaments. Depolymerization is produced by conformational changes in GDP-tubulin since microtubules assembled with a slowly hydrolizable analog of GTP, GMPCPP, are stable even at 20 mM Mg2+ or 5 mM

Topics: Animals; Antiviral Agents; Calcium; Guanosine Triphosphate; Magnesium; Microscopy, Phase-Contrast; Microscopy, Video; Microtubules; Monte Carlo Method; Protein Conformation; Tubulin

Dynamic instability of microtubules is characterized by stochastically alternating phases of growth and shrinkage and is hypothesized to be controlled by the conformation and nucleotide state of tubulin dimers within the microtubule lattice. Specifically, conformation changes (compression) in the tubulin dimer following the hydrolysis of GTP have been suggested to generate stress and drive depolymerization. In the present study, molecular dynamics simulations were used in tandem with in vitro experiments to investigate changes in depolymerization based on the presence of islands of uncompressed (GMPCPP) dimers in the microtubule lattice. Both methods revealed an exponential decay in the kinetic rate of depolymerization corresponding to the relative level of uncompressed (GMPCPP) dimers, beginning at approximately 20% incorporation. This slowdown was accompanied by a distinct morphological change from unpeeling "ram's horns" to blunt-ended dissociation at the microtubule end. Collectively these data demonstrated that islands of uncompressed dimers can alter the mechanism and kinetics of depolymerization in a manner consistent with promoting rescue events.

Topics: Animals; Guanosine Triphosphate; Hydrolysis; Kinetics; Microtubules; Molecular Dynamics Simulation; Polymerization; Protein Multimerization; Swine; Tubulin

Chloroplasts evolved from cyanobacterial endosymbiotic ancestors and their division is a complex process initiated by the assembly of cytoskeletal FtsZ (

Topics: Arabidopsis; Arabidopsis Proteins; Bacterial Proteins; Cell Division; Chloroplasts; Cytoskeleton; Eukaryotic Cells; Gene Expression Regulation, Plant; Guanosine Triphosphate; Kinetics; Nuclear Proteins; Pichia; Recombinant Proteins; Saccharomyces cerevisiae

Archaea and eukaryotes have ribosomal P stalks composed of anchor protein P0 and aP1 homodimers (archaea) or P1•P2 heterodimers (eukaryotes). These P stalks recruit translational GTPases to the GTPase-associated center in ribosomes to provide energy during translation. The C-terminus of the P stalk is known to selectively recognize GTPases. Here we investigated the interaction between the P stalk and elongation factor 2 by determining the structures of Pyrococcus horikoshii EF-2 (PhoEF-2) in the Apo-form, GDP-form, GMPPCP-form (GTP-form), and GMPPCP-form bound with 11 C-terminal residues of P1 (P1C11). Helical structured P1C11 binds to a hydrophobic groove between domain G and subdomain G' of PhoEF-2, where is completely different from that of aEF-1α in terms of both position and sequence, implying that such interaction characteristic may be requested by how GTPases perform their functions on the ribosome. Combining PhoEF-2 P1-binding assays with a structural comparison of current PhoEF-2 structures and molecular dynamics model of a P1C11-bound GDP form, the conformational changes of the P1C11-binding groove in each form suggest that in response to the translation process, the groove has three states: closed, open, and release for recruiting and releasing GTPases.

Topics: Amino Acid Sequence; Archaeal Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Peptide Elongation Factor 2; Protein Binding; Protein Conformation; Pyrococcus horikoshii; Ribosomal Proteins; Ribosomes; Sequence Homology, Amino Acid

Structures of wild-type K-Ras from crystals obtained in the presence of guanosine triphosphate (GTP) or its analogs have remained elusive. Of the K-Ras mutants, only K-RasG12D and K-RasQ61H are available in the PDB representing the activated form of the GTPase not in complex with other proteins. We present the crystal structure of wild-type K-Ras bound to the GTP analog GppCH

Topics: Catalytic Domain; Crystallography, X-Ray; Guanosine Triphosphate; Humans; Models, Molecular; Mutation; Protein Binding; Protein Conformation; Proto-Oncogene Proteins p21(ras); Proton Magnetic Resonance Spectroscopy

Membrane fission is a fundamental process in the regulation and remodelling of cell membranes. Dynamin, a large GTPase, mediates membrane fission by assembling around, constricting and cleaving the necks of budding vesicles

Topics: Biopolymers; Cell Membrane; Cryoelectron Microscopy; Dynamin I; Endocytosis; Guanosine Triphosphate; Humans; Hydrolysis; Models, Molecular; Mutant Proteins; Mutation; Protein Domains; Protein Multimerization

Microtubule (MT) is made of αβ-tubulin heterodimers that dynamically assemble into a hollow nanotube composed of straight protofilaments. MT dynamics is facilitated by hydrolysis of guanosine-5'-triphosphate (GTP) and can be inhibited by either anticancer agents like taxol or the nonhydrolyzable GTP analogues like GMPPCP. Using high-resolution synchrotron X-ray scattering, we have measured and analyzed the scattering curves from solutions of dynamic MT (in other words, in the presence of excess GTP and free of dynamic-inhibiting agents) and examined the effect of two MT stabilizers: taxol and GMPPCP. Previously, we have analyzed the structure of dynamic MT by docking the atomic model of tubulin dimer onto a 3-start left handed helical lattice, derived from the PDB ID 3J6F . 3J6F corresponds to a MT with 14 protofilaments. In this paper, we took into account the possibility of having MT structures containing between 12 and 15 protofilaments. MTs with 12 protofilaments were never observed. We determined the radii, the pitch, and the distribution of protofilament number that best fit the scattering data from dynamic MT or stabilized MT by taxol or GMPPCP. We found that the protofilament number distribution shifted when the MT was stabilized. Taxol increased the mass fraction of MT with 13 protofilaments and decreased the mass fraction of MT with 14 protofilaments. GMPPCP reduced the mass fraction of MT with 15 protofilaments and increased the mass fraction of MT with 14 protofilaments. The pitch, however, remained unchanged regardless of whether the MT was dynamic or stabilized. Higher tubulin concentrations increased the fraction of dynamic MT with 14 protofilaments.

Topics: Guanosine Triphosphate; Microscopy, Electron, Transmission; Microtubules; Molecular Docking Simulation; Paclitaxel; Protein Stability; Protein Structure, Quaternary; Scattering, Radiation; Tubulin; X-Rays

Cdc42 regulates pathways related to cell division. Dysregulation of Cdc42 can lead to cancer, cardiovascular diseases and neurodegenerative diseases. GTP induced activation mechanism plays an important role in the activity and biological functions of Cdc42. P-loop, Switch I and Switch II are critical regions modulating the enzymatic activity of Cdc42. We applied amide hydrogen/deuterium exchange coupled with liquid chromatography mass spectrometry (HDXMS) to investigate the dynamic changes of apo-Cdc42 after GDP, GTP and GMP-PCP binding. The natural substrate GTP induced significant decreases of deuteration in P-loop and Switch II, moderate changes of deuteration in Switch I and significant changes of deuteration in the α7 helix, a region far away from the active site. GTP binding induced similar effects on H/D exchange to its non-hydrolysable analog, GMP-PCP. HDXMS results indicate that GTP binding blocked the solvent accessibility in the active site leading to the decrease of H/D exchange rate surrounding the active site, and further triggered a conformational change resulting in the drastic decrease of H/D exchange rate at the remote α7 helix. Comparing the deuteration levels in three activation states of apo-Cdc42, Cdc42-GDP and Cdc42-GMP-PCP, the apo-Cdc42 has the most flexible structure, which can be stabilized by guanine nucleotide binding. The rates of H/D exchange of Cdc42-GDP are between the GMP-PCP-bound and the apo form, but more closely to the GMP-PCP-bound form. Our results show that the activation of Cdc42 is a process of conformational changes involved with P-loop, Switch II and α7 helix for structural stabilization.

Topics: Amino Acid Sequence; cdc42 GTP-Binding Protein; Deuterium Exchange Measurement; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Models, Molecular; Molecular Sequence Data; Peptides; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Static Electricity

Using single-molecule fluorescence microscopy, we investigated the dynamics of dye-labeled EB1, a +TIP microtubule binding protein. To promote EB1 binding along the entire microtubule length, we formed microtubules using the nonhydrolyzable GTP analogs GMPCPP and GTPγS. Through precise tracking of the motions of individual dye-labeled proteins, we found EB1 to be highly dynamic and continuously diffusive while bound to a microtubule, with a diffusion coefficient and characteristic binding lifetime that were sensitive to both the choice of GTP analog and the buffer ionic strength. Using fluorescence-based equilibrium binding measurements, we found EB1 binding to be cooperative and also sensitive to GTP analog and ionic strength. By tracking the motion of a small number of individually-labeled EB1 proteins within a bath of unlabeled EB1 proteins, we determined the effects of increasing the total EB1 concentration on binding and dynamics. We found that the diffusion coefficient decreased with increasing EB1 concentration, which may be due at least in part, to the cooperativity of EB1 binding. Our results may have important consequences for the assembly and organization of the growing microtubule plus-end.

Topics: Guanosine Triphosphate; Humans; Microscopy, Fluorescence; Microtubule-Associated Proteins; Microtubules; Osmolar Concentration; Protein Binding

The dynamic regulation of microtubules (MTs) during mitosis is critical for accurate chromosome segregation and genome stability. Cancer cell lines with hyperstabilized kinetochore MTs have increased segregation errors and elevated chromosomal instability (CIN), but the genetic defects responsible remain largely unknown. The MT depolymerase MCAK (mitotic centromere-associated kinesin) can influence CIN through its impact on MT stability, but how its potent activity is controlled in cells remains unclear. In this study, we show that GTSE1, a protein found overexpressed in aneuploid cancer cell lines and tumors, regulates MT stability during mitosis by inhibiting MCAK MT depolymerase activity. Cells lacking GTSE1 have defects in chromosome alignment and spindle positioning as a result of MT instability caused by excess MCAK activity. Reducing GTSE1 levels in CIN cancer cell lines reduces chromosome missegregation defects, whereas artificially inducing GTSE1 levels in chromosomally stable cells elevates chromosome missegregation and CIN. Thus, GTSE1 inhibition of MCAK activity regulates the balance of MT stability that determines the fidelity of chromosome alignment, segregation, and chromosomal stability.

Topics: Anaphase; Cell Line, Tumor; Chromosomal Instability; Chromosome Segregation; Chromosomes, Human; Guanosine Triphosphate; Humans; Kinesins; Kinetochores; Microtubule-Associated Proteins; Microtubules; Mitosis; Protein Binding; Spindle Apparatus

The microtubule (MT)-kinesin biomolecular motor system has attracted considerable attention due to its possible applications in artificial biomachines. Recently, an active self-organization (AcSO) method has been established to integrate MT filaments into highly organized assembled structures. The ring-shaped MT assembly, one of the structures derived from the AcSO of MTs, can convert the translational motion of MTs into rotational motion. Due to this attractive feature, the ring-shaped MT assembly appears to be a promising candidate for developing artificial devices and for future nanotechnological applications. In this work, we have investigated the effect of length and rigidity of the MT filaments on the size of the ring-shaped MT assembly in the AcSO process. We show that the size of the ring-shaped MT assembly can be controlled by tuning the length and rigidity of MT filaments employed in the AcSO. Longer and stiffer MT filaments led to larger ring-shaped assemblies through AcSO, whereas AcSO of shorter and less stiff MT filaments produced smaller ring-shaped assemblies. This work might be important for the development of biomolecular motor based artificial biomachines, especially where size control of ring-shaped MT assembly will play an important role.

Topics: Animals; Biomimetics; Guanosine Triphosphate; Kinesins; Microscopy, Fluorescence; Microtubules; Swine

The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state microtubules over GDP-state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide-free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide-free KIF5C and the cryo-electron microscopic structure of nucleotide-free KIF5C complexed with the GTP-state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP-state microtubule and KIF5C. KIF5C acquires the 'rigor conformation', where mobile switches I and II are stabilized through L11 and the initial portion of the neck-linker, facilitating effective ADP release and the weak-to-strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP-state microtubule in a similar manner to GDP-taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP-state microtubules.

Topics: Adenosine Diphosphate; Amino Acid Sequence; Animals; Binding Sites; Cryoelectron Microscopy; Crystallography, X-Ray; Guanosine Triphosphate; Kinesins; Mice; Microtubules; Models, Molecular; Molecular Docking Simulation; Molecular Sequence Data; Protein Conformation

Microtubules are born and reborn continuously, even during quiescence. These polymers are nucleated from templates, namely γ-tubulin ring complexes (γ-TuRCs) and severed microtubule ends. Using single-molecule biophysics, we show that nucleation from γ-TuRCs, axonemes and seed microtubules requires tubulin concentrations that lie well above the critical concentration. We measured considerable time lags between the arrival of tubulin and the onset of steady-state elongation. Microtubule-associated proteins (MAPs) alter these time lags. Catastrophe factors (MCAK and EB1) inhibited nucleation, whereas a polymerase (XMAP215) and an anti-catastrophe factor (TPX2) promoted nucleation. We observed similar phenomena in cells. We conclude that GTP hydrolysis inhibits microtubule nucleation by destabilizing the nascent plus ends required for persistent elongation. Our results explain how MAPs establish the spatial and temporal profile of microtubule nucleation.

Topics: Animals; Axoneme; Cell Line, Tumor; Centrosome; CHO Cells; Cricetinae; Cricetulus; Green Fluorescent Proteins; Guanosine Triphosphate; Humans; Hydrolysis; Immunoblotting; Kinetics; LLC-PK1 Cells; Microscopy, Electron; Microscopy, Fluorescence; Microtubule-Associated Proteins; Microtubules; Nocodazole; Polymerization; Swine; Tubulin; Tubulin Modulators

GTP cyclohydrolase II (GCHII), catalyzes the conversion of GTP to 2,5-diamino-6-β-ribosyl-4(3H)-pyrimidinone-5'-phosphate and has been shown to be essential for pathogens. Here we describe the biochemical, kinetic and structural characterization of GCHII from Helicobacter pylori (hGCHII). The crystal structure of hGCHII, unlike other GCHII structures, revealed that cysteines at the active site existed in oxidized state forming two disulfide bonds and lacked Zn(2+) that was shown to be indispensable for catalytic activity in other species. However, incubation of hGCHII with hydrogen peroxide, an oxidizing agent, followed by PAR-assay showed that Zn(2+) was intrinsically present, indicating that all cysteines at the catalytic site remained in reduced state. Moreover, site directed mutagenesis of catalytic site cysteines revealed that only three, out of four cysteines were essential for hGCHII activity. These results, though, indicated that hGCHII crystallized in oxidized form, the expulsion of Zn(2+) upon oxidation of catalytic cysteines revealed its ability to act in response to the redox environment. Exploring further, incubation of hGCHII with reversible thiol modifying agent S-methyl-methane-thiosulfonate resulted in loss of GCHII activity due to oxidation of its cysteine residues as revealed by mass spectrometry studies. However, addition of reducing agent DTT partially restored the hGCHII catalytic activity. Taken together, these results demonstrate that hGCHII can regulate its catalytic activity depending on the redox environment, a function hitherto unknown for GCHII.

Topics: Amino Acid Sequence; Bacterial Proteins; Catalytic Domain; Conserved Sequence; Crystallography, X-Ray; GTP Cyclohydrolase; Guanosine Triphosphate; Helicobacter pylori; Kinetics; Models, Molecular; Molecular Sequence Data; Oxidation-Reduction; Peptide Fragments; Protein Binding; Protein Structure, Secondary

Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe.

Topics: Animals; Guanosine Diphosphate; Guanosine Triphosphate; Kinesins; Microtubule-Associated Proteins; Microtubules; Models, Biological; Molecular Conformation; Protein Multimerization; Protein Transport; Rats; Schizosaccharomyces pombe Proteins; Surface Properties; Sus scrofa; Tubulin

It has been demonstrated that Tau exists on the microtubule lattice in both diffusing and static populations, but how this may relate to Tau function is currently unclear. Tau isoforms are developmentally regulated and have been shown to have disparate effects on microtubule polymerization, the ability to bind microtubules, and the ability to inhibit kinesin. It has also been shown that Tau is sensitive to microtubule stabilizing agents and the ability to affect the persistence length of microtubules and to inhibit kinesin can be altered by stabilizing microtubules with various nucleotide analogs. Given these observations, it is likely the behavior of Tau is dictated by both the isoform of Tau and by structural changes in the microtubule lattice. In the present study, we use single molecule imaging to examine the behavior of the three-repeat short (3RS) isoform and the four-repeat long (4RL) isoform on different microtubule tracks stabilized with either paclitaxel or guanylyl-(α,β)-methylene-diphosphate (GMPCPP). On paclitaxel-stabilized microtubules, we find 3RS-Tau favors the static conformation and forms complexes consisting of 2-3 molecules, while 4RL-Tau predominantly exists as a single molecule equally distributed between the static and diffusing populations. However, on GMPCPP-stabilized microtubules both isoforms favor the diffusing conformation and do not form static complexes composed of more than one Tau molecule. We find both isoforms of Tau interconvert between static and diffusing populations on the microtubule surface, and the equilibrium between these two states depends on both the isoform of Tau and the structure of the underlying microtubule lattice.

Topics: Animals; Cattle; Diffusion; Guanosine Triphosphate; Humans; Kymography; Microtubules; Paclitaxel; Protein Isoforms; tau Proteins; Time Factors

The neck-linker is a structurally conserved region among most members of the kinesin superfamily of molecular motor proteins that is critical for kinesin's processive transport of intracellular cargo along the microtubule surface. Variation in the neck-linker length has been shown to directly modulate processivity in different kinesin families; for example, kinesin-1, with a shorter neck-linker, is more processive than kinesin-2. Although small differences in processivity are likely obscured in vivo by the coupling of most cargo to multiple motors, longer and more flexible neck-linkers may allow different kinesins to navigate more efficiently around the many obstacles, including microtubule-associated proteins (MAPs), that are found on the microtubule surface within cells. We hypothesize that, due to its longer neck-linker, kinesin-2 can more easily navigate obstacles (e.g., MAPs) on the microtubule surface than kinesin-1. We used total internal reflection fluorescence microscopy to observe single-molecule motility from different kinesin-1 and kinesin-2 neck-linker chimeras stepping along microtubules in the absence or presence of two Tau isoforms, 3RS-Tau and 4RL-Tau, both of which are MAPs that are known to differentially affect kinesin-1 motility. Our results demonstrate that unlike kinesin-1, kinesin-2 is insensitive to the presence of either Tau isoform, and appears to have the ability to switch protofilaments while stepping along the microtubule when challenged by an obstacle, such as Tau. Thus, although kinesin-1 may be more processive, the longer neck-linker length of kinesin-2 allows it to be better optimized to navigate the complex microtubule landscape. These results provide new insight, to our knowledge, into how kinesin-1 and kinesin-2 may work together for the efficient delivery of cargo in cells.

Topics: Amino Acid Sequence; Animals; Cattle; Drosophila melanogaster; Guanosine Triphosphate; Kinesins; Kymography; Mice; Microtubules; Molecular Sequence Data; Paclitaxel; Protein Structure, Tertiary; Protein Transport; Structure-Activity Relationship; tau Proteins

Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is inhibited by anticancer agents like Taxol. We provide insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7-5.6 Å) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α-tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α- and β-tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces.

Topics: Animals; Cryoelectron Microscopy; Crystallography, X-Ray; Guanosine Triphosphate; Humans; Hydrolysis; Microtubules; Models, Molecular; Paclitaxel; Protein Conformation; Tubulin

Microtubules are cytoskeletal filaments responsible for cell morphology and intracellular organization. Their dynamical and mechanical properties are regulated through the nucleotide state of the tubulin dimers and the binding of drugs and/or microtubule-associated proteins. Interestingly, microtubule-stabilizing factors have differential effects on microtubule mechanics, but whether stabilizers have cumulative effects on mechanics or whether one effect dominates another is not clear. This is especially important for the chemotherapeutic drug Taxol, an important anticancer agent and the only known stabilizer that reduces the rigidity of microtubules. First, we ask whether Taxol will combine additively with another stabilizer or whether one stabilizer will dominate another. We call microtubules in the presence of Taxol and another stabilizer, doubly stabilized. Second, since Taxol is often added to a number of cell types for therapeutic purposes, it is important from a biomedical perspective to understand how Taxol added to these systems affects the mechanical properties in treated cells. To address these questions, we use the method of freely fluctuating filaments with our recently developed analysis technique of bootstrapping to determine the distribution of persistence lengths of a large population of microtubules treated with different stabilizers, including Taxol, guanosine-5' [(α, β)-methyleno] triphosphate, guanosine-5'-O-(3-thiotriphosphate), tau, and MAP4. We find that combinations of these stabilizers have novel effects on the mechanical properties of microtubules.

Topics: Animals; Biomechanical Phenomena; Fourier Analysis; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Mechanical Phenomena; Microtubule-Associated Proteins; Microtubules; Normal Distribution; Paclitaxel; Protein Multimerization; Protein Structure, Quaternary; Swine; tau Proteins; Temperature

Single-axis cryo-electron tomography of vitrified specimens has become a method of choice to reconstruct in three dimensions macromolecular assemblies in their cellular context or prepared from purified components. Here, we asked how a dual-axis acquisition scheme would improve three-dimensional reconstructions of microtubules assembled in vitro. We show that in single-axis tomograms, microtubules oriented close to the perpendicular of the tilt axis display diminished contrast, and ultimately transform into sets of parallel lines oriented in the direction of the electron beam when observed in cross-section. Analysis of their three-dimensional Fourier transform indicates that this imaging artifact is due to a decrease in the angular sampling of their equatorial components. Although the second orthogonal series does not fully complement the first one at the specimen level due to increased radiation damage, it still allows elongated features oriented in any directions to be correctly reconstructed, which might be essential for highly heterogeneous specimens such as cells.

Topics: Cryoelectron Microscopy; Fourier Analysis; Guanosine Triphosphate; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Microtubules; Tomography, X-Ray Computed

Microtubules are dynamic polymers of GTP- and GDP-tubulin that undergo stochastic transitions between growing and shrinking phases. Rescues, the conversion from shrinking to growing, have recently been proposed to be to the result of regrowth at GTP-tubulin islands within the lattice of growing microtubules. By introducing mixed GTP/GDP/GMPCPP (GXP) regions within the lattice of dynamic microtubules, we reconstituted GXP islands in vitro (GMPCPP is the slowly hydrolyzable GTP analog guanosine-5'-[(α,β)-methyleno]triphosphate). We found that such islands could reproducibly induce rescues and that the probability of rescue correlated with both the size of the island and the percentage of GMPCPP-tubulin within the island. The islands slowed the depolymerization rate of shortening microtubules and promoted regrowth more readily than GMPCPP seeds. Together, these findings provide new mechanistic insights supporting the possibility that rescues could be triggered by enriched GTP-tubulin regions and present a new tool for studying such rescue events in vitro.

Topics: Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Microscopy, Fluorescence; Microtubules; Molecular Dynamics Simulation; Polymers; Tubulin

The kinesins-8 were originally thought to be microtubule depolymerases, but are now emerging as more versatile catalysts of microtubule dynamics. We show here that S. pombe Klp5-436 and Klp6-440 are non-processive plus-end-directed motors whose in vitro velocities on S. pombe microtubules at 7 and 23 nm s(-1) are too slow to keep pace with the growing tips of dynamic interphase microtubules in living S. pombe. In vitro, Klp5 and 6 dimers exhibit a hitherto-undescribed combination of strong enhancement of microtubule nucleation with no effect on growth rate or catastrophe frequency. By contrast in vivo, both Klp5 and Klp6 promote microtubule catastrophe at cell ends whilst Klp6 also increases the number of interphase microtubule arrays (IMAs). Our data support a model in which Klp5/6 bind tightly to free tubulin heterodimers, strongly promoting the nucleation of new microtubules, and then continue to land as a tubulin-motor complex on the tips of growing microtubules, with the motors then dissociating after a few seconds residence on the lattice. In vivo, we predict that only at cell ends, when growing microtubule tips become lodged and their growth slows down, will Klp5/6 motor activity succeed in tracking growing microtubule tips. This mechanism would allow Klp5/6 to detect the arrival of microtubule tips at cells ends and to amplify the intrinsic tendency for microtubules to catastrophise in compression at cell ends. Our evidence identifies Klp5 and 6 as spatial regulators of microtubule dynamics that enhance both microtubule nucleation at the cell centre and microtubule catastrophe at the cell ends.

Topics: Adenosine Triphosphatases; Animals; Enzyme-Linked Immunosorbent Assay; Gene Deletion; Guanosine Triphosphate; Kinesins; Microspheres; Microtubule-Associated Proteins; Microtubules; Models, Biological; Multiprotein Complexes; Protein Multimerization; Protein Stability; Protein Transport; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Sus scrofa; Time Factors; Tubulin

The concerted formation of a narrow distribution of oligomeric FtsZ species in the presence of GTP or a GTP analogue under close to physiological conditions (neutral pH and 0.5 M K(+)) has been characterized recently by various biophysical methods [Monterroso, B., et al. (2012) Biochemistry 51, 4541-4550]. An equilibrium model may semiquantitatively account for the results of this study; in the model, FtsZ self-associates in a noncooperative fashion to form linear fibrils, that upon increasing to a certain size exhibit an increasing tendency to form closed cyclic fibrils, as previously suggested [González, J. M., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 1895-1900]. The closed cyclic fibrils are formed when the natural curvature and flexibility of a linear oligomer bring the ends of a linear fiber sufficiently close to overcome the entropic barrier to loop closure. The size distribution of cyclic oligomers is thus a reflection of the tendency toward curvature of linear fibrils of FtsZ under the conditions used in these experiments.

Topics: Cytoskeletal Proteins; Dose-Response Relationship, Drug; Guanosine Triphosphate; Light; Magnesium; Models, Biological; Protein Multimerization; Protein Structure, Secondary; Rotation; Scattering, Radiation

Microtubules are dynamic polymers that stochastically switch between growing and shrinking phases. Microtubule dynamics are regulated by guanosine triphosphate (GTP) hydrolysis by β-tubulin, but the mechanism of this regulation remains elusive because high-resolution microtubule structures have only been revealed for the guanosine diphosphate (GDP) state. In this paper, we solved the cryoelectron microscopy (cryo-EM) structure of microtubule stabilized with a GTP analogue, guanylyl 5'-α,β-methylenediphosphonate (GMPCPP), at 8.8-Å resolution by developing a novel cryo-EM image reconstruction algorithm. In contrast to the crystal structures of GTP-bound tubulin relatives such as γ-tubulin and bacterial tubulins, significant changes were detected between GMPCPP and GDP-taxol microtubules at the contacts between tubulins both along the protofilament and between neighboring protofilaments, contributing to the stability of the microtubule. These findings are consistent with the structural plasticity or lattice model and suggest the structural basis not only for the regulatory mechanism of microtubule dynamics but also for the recognition of the nucleotide state of the microtubule by several microtubule-binding proteins, such as EB1 or kinesin.

Topics: Algorithms; Animals; Brain; Cryoelectron Microscopy; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Image Processing, Computer-Assisted; Microtubules; Models, Molecular; Molecular Conformation; Paclitaxel; Protein Conformation; Stochastic Processes; Swine; Tubulin

Cotranslational targeting of membrane and secretory proteins is mediated by the universally conserved signal recognition particle (SRP). Together with its receptor (SR), SRP mediates the guanine triphosphate (GTP)-dependent delivery of translating ribosomes bearing signal sequences to translocons on the target membrane. Here, we present the crystal structure of the SRP:SR complex at 3.9 angstrom resolution and biochemical data revealing that the activated SRP:SR guanine triphosphatase (GTPase) complex binds the distal end of the SRP hairpin RNA where GTP hydrolysis is stimulated. Combined with previous findings, these results suggest that the SRP:SR GTPase complex initially assembles at the tetraloop end of the SRP RNA and then relocalizes to the opposite end of the RNA. This rearrangement provides a mechanism for coupling GTP hydrolysis to the handover of cargo to the translocon.

Topics: Bacterial Proteins; Base Sequence; Binding Sites; Crystallization; Crystallography, X-Ray; Enzyme Activation; Escherichia coli; Escherichia coli Proteins; GTP Phosphohydrolases; Guanosine Triphosphate; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Models, Biological; Models, Molecular; Nucleic Acid Conformation; Protein Conformation; Protein Multimerization; Protein Structure, Tertiary; Protein Transport; Receptors, Cytoplasmic and Nuclear; Ribosomal Proteins; Ribosomes; RNA, Bacterial; Signal Recognition Particle

Microtubule (MT) length and location is tightly controlled in cells. One novel family of MT-associated proteins that regulates MT dynamics is the MT-severing enzymes. In this work, we investigate how katanin (p60), believed to be the first discovered severing enzyme, binds and severs MTs via single molecule total internal reflection fluorescence microscopy. We find that severing activity depends on katanin concentration. We also find that katanin can remove tubulin dimers from the ends of MTs, appearing to depolymerize MTs. Strikingly, katanin localizes and severs at the interface of GMPCPP-tubulin and GDP-tubulin suggesting that it targets to protofilament-shift defects. Finally, we observe that binding duration, mobility, and oligomerization are ATP dependent.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Baculoviridae; Drosophila melanogaster; Drosophila Proteins; Fluorescence; Green Fluorescent Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Katanin; Microtubules; Models, Biological; Paclitaxel; Photobleaching; Polymerization; Protein Binding; Protein Structure, Quaternary; Protein Structure, Tertiary; Protein Transport; Recombinant Fusion Proteins; Tubulin

Polarized transport in neurons is fundamental for the formation of neuronal circuitry. A motor domain-containing truncated KIF5 (a kinesin-1) recognizes axonal microtubules, which are enriched in EB1 binding sites, and selectively accumulates at the tips of axons. However, it remains unknown what cue KIF5 recognizes to result in this selective accumulation. We found that axonal microtubules were preferentially stained by the anti-GTP-tubulin antibody hMB11. Super-resolution microscopy combined with EM immunocytochemistry revealed that hMB11 was localized at KIF5 attachment sites. In addition, EB1, which binds preferentially to guanylyl-methylene-diphosphate (GMPCPP) microtubules in vitro, recognized hMB11 binding sites on axonal microtubules. Further, expression of hMB11 antibody in neurons disrupted the selective accumulation of truncated KIF5 in the axon tips. In vitro studies revealed approximately threefold stronger binding of KIF5 motor head to GMPCPP microtubules than to GDP microtubules. Collectively, these data suggest that the abundance of GTP-tubulin in axonal microtubules may underlie selective KIF5 localization and polarized axonal vesicular transport.

Topics: Animals; Antibodies; Axons; Biological Transport; Guanosine Triphosphate; Kinesins; Mice; Microtubule-Associated Proteins; Microtubules; Swine; Tubulin

Leucine rich repeat kinase 2 (LRRK2) is a Parkinson's disease (PD) gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC) GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

Topics: 14-3-3 Proteins; Amino Acid Substitution; Binding Sites; GTP Phosphohydrolases; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; HEK293 Cells; Humans; Immunoblotting; Immunoprecipitation; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mutation; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Recombinant Proteins

We have analyzed the substrate kinetics of the GTPase activity of FtsZ and the effects of two different GTPase inhibitors, GDP and the slowly hydrolyzable GTP analogue GMPCPP. In the absence of inhibitors the GTPase activity follows simple Michaelis-Menten kinetics, and both GDP and GMPCPP inhibited the activity in a competitive manner. These results indicate that the GTPase active sites in FtsZ filaments are independent of each other, a feature relevant to elucidate the role of GTP hydrolysis in FtsZ function and cell division.

Topics: Bacterial Proteins; Binding, Competitive; Catalytic Domain; Cell Division; Cytoskeletal Proteins; Enzyme Inhibitors; Escherichia coli; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Kinetics

By using rigid microtubules (MTs) prepared by polymerization with guanylyl-(alpha, beta)-methylene-diphosphonate GMPCPP, giant straight-shaped MT bundles were selectively obtained through a dynamic self-assembly process. We demonstrate the effect of the rigidity on the shape and motility of MT bundle composed of GMPCPP-polymerized MTs (GMPCPP-MTs) compared with control MTs that were polymerized with GTP and stabilized with paclitaxel.

Topics: Animals; Guanosine Triphosphate; Kinesins; Microtubules; Movement; Swine; Tubulin

The de novo initiating RNA-directed RNA polymerase (RdRP), P2, forms the central machinery in the infection cycle of the bacteriophage phi6 by performing the dual tasks of replication and transcription of the double-stranded RNA genome in the host cell. By measurement and quantitative analysis of multiple-quantum spin-relaxation data for the delta1 positions of Ile residues that are distributed over the 3D-fold of P2, we find that the enzyme is dynamic both on the fast (ps-ns) and slow (micros-ms) timescales. The characteristics of several motional modes including those that coincide with the catalytic timescale (500-800/s) are altered in the presence of substrate analogs and single-stranded RNA templates. These studies reveal the plasticity of this finely tuned molecular machine and represent a first step towards linking structural information available from a host of crystal structures to catalytic mechanisms and timescales obtained from the measurements of kinetics for homologous systems in solution.

Topics: Bacteriophage phi 6; Guanosine Triphosphate; Isoleucine; Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; RNA; RNA-Dependent RNA Polymerase

Kinetic measurements of enzyme activity indicate that type I pantothenate kinase from Mycobacterium tuberculosis has dual substrate specificity for ATP and GTP, unlike the enzyme from Escherichia coli, which shows a higher specificity for ATP. A molecular explanation for the difference in the specificities of the two homologous enzymes is provided by the crystal structures of the complexes of the M. tuberculosis enzyme with (1) GMPPCP and pantothenate, (2) GDP and phosphopantothenate, (3) GDP, (4) GDP and pantothenate, (5) AMPPCP, and (6) GMPPCP, reported here, and the structures of the complexes of the two enzymes involving coenzyme A and different adenyl nucleotides reported earlier. The explanation is substantially based on two critical substitutions in the amino acid sequence and the local conformational change resulting from them. The structures also provide a rationale for the movement of ligands during the action of the mycobacterial enzyme. Dual specificity of the type exhibited by this enzyme is rare. The change in locations of ligands during action, observed in the case of the M. tuberculosis enzyme, is unusual, so is the striking difference between two homologous enzymes in the geometry of the binding site, locations of ligands, and specificity. Furthermore, the dual specificity of the mycobacterial enzyme appears to have been caused by a biological necessity.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Ligands; Models, Molecular; Molecular Sequence Data; Mycobacterium tuberculosis; Pantothenic Acid; Phosphotransferases (Alcohol Group Acceptor); Protein Structure, Tertiary; Substrate Specificity

FtsZ is an essential bacterial GTPase that polymerizes at midcell, recruits the division machinery, and may generate constrictive forces necessary for cytokinesis. However, many of the mechanistic details underlying these functions are unknown. We sought to identify FtsZ-binding proteins that influence FtsZ function in Caulobacter crescentus. Here, we present a microscopy-based screen through which we discovered two FtsZ-binding proteins, FzlA and FzlC. FzlA is conserved in α-proteobacteria and was found to be functionally critical for cell division in Caulobacter. FzlA altered FtsZ structure both in vivo and in vitro, forming stable higher-order structures that were resistant to depolymerization by MipZ, a spatial determinant of FtsZ assembly. Electron microscopy revealed that FzlA organizes FtsZ protofilaments into striking helical bundles. The degree of curvature induced by FzlA depended on the nucleotide bound to FtsZ. Induction of FtsZ curvature by FzlA carries implications for regulating FtsZ function by modulating its superstructure.

Topics: Adenosine Triphosphatases; Bacterial Outer Membrane Proteins; Bacterial Proteins; Caulobacter crescentus; Cell Cycle; Cell Division; Cell Shape; Cytoskeletal Proteins; Gene Expression; Guanosine Diphosphate; Guanosine Triphosphate; Microscopy, Electron, Transmission; Microscopy, Interference; N-Acetylglucosaminyltransferases; Polymerization; Protein Binding; Protein Interaction Mapping; Protein Multimerization; Protein Structure, Quaternary

Because microtubules are the structural elements of cells, it is essential to study the mechanical properties of single microtubules under physiological conditions. Previously, we measured the effect of temperature on the flexural rigidity of a single taxol-stabilized microtubule and found that the flexural rigidity is 2.5×10(-24)Nm(2), independent of temperature in the 20-35°C range. Employing the same technique here, we have measured the flexural rigidity of microtubules polymerized in the presence of guanylyl-(a,b)-methylene-diphosphonate (GMPCPP, the slowly hydrolyzable GTP analogue) and in the presence of GTP only; both of the states were taxol-free. The obtained values were approximately 5-fold (for GMPCPP) and three- to 4-fold (for GTP) greater than those of taxol-stabilized microtubules. Interestingly, rigidity decreased as temperature increased, that is, temperature dependence was only observed in taxol-free microtubules. Length dependence was also observed. These results indicate that the transition of microtubule's rigidity is associated with the tubulin conformation change from a GTP-bound state to a GDP-bound state in the α/β subunit. We discuss the relationship of the regulation mechanism of the microtubules in the cell body to the changes in rigidity through hydrolysis.

Topics: Animals; Guanosine Triphosphate; Microtubules; Paclitaxel; Protein Conformation; Swine; Temperature; Tubulin; Tubulin Modulators

Fission yeast expresses two kinesin-8s, previously identified and characterized as products of the klp5(+) and klp6(+) genes. These polypeptides colocalize throughout the vegetative cell cycle as they bind cytoplasmic microtubules during interphase, spindle microtubules, and/or kinetochores during early mitosis, and the interpolar spindle as it elongates in anaphase B. Here, we describe in vitro properties of these motor proteins and some truncated versions expressed in either bacteria or Sf9 cells. The motor-plus-neck domain of Klp6p formed soluble dimers that cross-linked microtubules and showed both microtubule-activated ATPase and plus-end-directed motor activities. Full-length Klp5p and Klp6p, coexpressed in Sf9 cells, formed soluble heterodimers with the same activities. The latter recombinant protein could also couple microbeads to the ends of shortening microtubules and use energy from tubulin depolymerization to pull a load in the minus end direction. These results, together with the spindle localizations of these proteins in vivo and their requirement for cell viability in the absence of the Dam1/DASH kinetochore complex, support the hypothesis that fission yeast kinesin-8 contributes both to chromosome congression to the metaphase plate and to the coupling of spindle microtubules to kinetochores during anaphase A.

Topics: Adenosine Triphosphatases; Alleles; Biological Transport; Cross-Linking Reagents; Genes, Fungal; Guanosine Triphosphate; Kinesins; Microspheres; Microtubule-Associated Proteins; Microtubules; Molecular Motor Proteins; Mutant Proteins; Paclitaxel; Protein Binding; Protein Multimerization; Rotation; Schizosaccharomyces; Schizosaccharomyces pombe Proteins

Dynamin-related proteins (DRPs) can generate forces to remodel membranes. In cells, DRPs require additional proteins [DRP-associated proteins (DAPs)] to conduct their functions. To dissect the mechanistic role of a DAP, we used the yeast mitochondrial division machine as a model, which requires the DRP Dnm1, and two other proteins, Mdv1 and Fis1. Mdv1 played a postmitochondrial targeting role in division by specifically interacting and coassembling with the guanosine triphosphate-bound form of Dnm1. This regulated interaction nucleated and promoted the self-assembly of Dnm1 into helical structures, which drive membrane scission. The nucleation of DRP assembly probably represents a general regulatory strategy for this family of filament-forming proteins, similar to F-actin regulation.

Topics: Adaptor Proteins, Signal Transducing; GTP Phosphohydrolases; Guanosine Triphosphate; Intracellular Membranes; Kinetics; Liposomes; Mitochondria; Mitochondrial Proteins; Models, Biological; Protein Binding; Protein Conformation; Protein Structure, Secondary; Saccharomyces cerevisiae Proteins

Individual dynamic microtubules can generate pushing or pulling forces when their growing or shrinking ends are in contact with cellular objects such as the cortex or chromosomes. These microtubules can operate in parallel bundles, for example when interacting with mitotic chromosomes. Here, we investigate the force-generating capabilities of a bundle of growing microtubules and study the effect that force has on the cooperative dynamics of such a bundle. We used an optical tweezers setup to study microtubule bundles growing against a microfabricated rigid barrier in vitro. We show that multiple microtubules can generate a pushing force that increases linearly with the number of microtubules present. In addition, the bundle can cooperatively switch to a shrinking state, due to a force-induced coupling of the dynamic instability of single microtubules. In the presence of GMPCPP, bundle catastrophes no longer occur, and high bundle forces are reached more effectively. We reproduce the observed behavior with a simple simulation of microtubule bundle dynamics that takes into account previously measured force effects on single microtubules. Using this simulation, we also show that a constant compressive force on a growing bundle leads to oscillations in bundle length that are of potential relevance for chromosome oscillations observed in living cells.

Topics: Animals; Biomechanical Phenomena; Computer Simulation; Guanosine Triphosphate; Microtubules; Sea Urchins

Microtubules are significant therapeutic targets for the treatment of cancer, where suppression of microtubule dynamicity by drugs such as paclitaxel forms the basis of clinical efficacy. Peloruside A, a macrolide isolated from New Zealand marine sponge Mycale hentscheli, is a microtubule-stabilizing agent that synergizes with taxoid drugs through a unique site and is an attractive lead compound in the development of combination therapies. We report here unique allosteric properties of microtubule stabilization via peloruside A and present a structural model of the peloruside-binding site. Using a strategy involving comparative hydrogen-deuterium exchange mass spectrometry of different microtubule-stabilizing agents, we suggest that taxoid-site ligands epothilone A and docetaxel stabilize microtubules primarily through improved longitudinal interactions centered on the interdimer interface, with no observable contributions from lateral interactions between protofilaments. The mode by which peloruside A achieves microtubule stabilization also involves the interdimer interface, but includes contributions from the alpha/beta-tubulin intradimer interface and protofilament contacts, both in the form of destabilizations. Using data-directed molecular docking simulations, we propose that peloruside A binds within a pocket on the exterior of beta-tubulin at a previously unknown ligand site, rather than on alpha-tubulin as suggested in earlier studies.

Topics: Allosteric Regulation; Amino Acid Sequence; Animals; Antineoplastic Agents, Phytogenic; Binding Sites; Bridged Bicyclo Compounds, Heterocyclic; Cattle; Dimerization; Guanosine Triphosphate; Lactones; Ligands; Mass Spectrometry; Microtubules; Models, Molecular; Molecular Sequence Data; Peptides; Protein Isoforms; Protein Structure, Quaternary; Sequence Alignment; Tubulin; Tubulin Modulators

Atomic force microscopy (AFM) has been used to investigate the local mechanical and structural properties of microtubules polymerized using guanylyl-alpha-beta-methylene diphosphonate (GMPCPP), a slowly hydrolyzable analogue of guanosine triphosphate. Using a combination of AFM imaging and local force spectroscopy, GMPCPP-polymerized microtubules have been qualitatively and quantitatively compared to paclitaxel-stabilized microtubules. GMPCPP-polymerized microtubules qualitatively display a greater resistance to destruction by the AFM probe tip during imaging and during deformation measurements and maintain structural details after indentation. In addition, using force spectroscopy taken during the indentation and collapse of individual microtubules with the AFM probe tip, an effective spring constant of the microtubule wall (kMT) for both types of microtubules was determined. The average kMT of GMPCPP-polymerized microtubules, 0.172 N/m, is more than twice that of paclitaxel-stabilized microtubules. These results complement previously reported measurements of bending experiments on GMPCPP-polymerized and paclitaxel-stabilized microtubules.

Topics: Antineoplastic Agents; Biopolymers; Guanosine Triphosphate; Hydrolysis; Microscopy, Atomic Force; Microtubules; Paclitaxel

The application of the (31)P NMR spectroscopy to large proteins or protein complexes in solution is hampered by a relatively low intrinsic sensitivity coupled with large line widths. Therefore, the assignment of the phosphorus signals by two-dimensional NMR methods in solution is often extremely time consuming. In contrast, the quality of solid-state NMR spectra is not dependent on the molecular mass and the solubility of the protein. For the complex of Ras with the GTP-analogue GppCH(2)p we show solid-state (31)P NMR methods to be more sensitive by almost one order of magnitude than liquid-state NMR. Thus, solid-state NMR seems to be the method of choice for obtaining the resonance assignment of the phosphorus signals of protein complexes in solution. Experiments on Ras.GDP complexes show that the microcrystalline sample can be substituted by a precipitate of the sample and that unexpectedly the two structural states observed earlier in solution are present in crystals as well.

Topics: Crystallization; Guanosine Triphosphate; Molecular Weight; Nuclear Magnetic Resonance, Biomolecular; Phosphorus Isotopes; Proteins; ras Proteins

The signal recognition particle (SRP) GTPases Ffh and FtsY play a central role in co-translational targeting of proteins, assembling in a GTP-dependent manner to generate the SRP targeting complex at the membrane. A suite of residues in FtsY have been identified that are essential for the hydrolysis of GTP that accompanies disengagement. We have argued previously on structural grounds that this region mediates interactions that serve to activate the complex for disengagement and term it the activation region. We report here the structure of a complex of the SRP GTPases formed in the presence of GDP:AlF4. This complex accommodates the putative transition-state analog without undergoing significant change from the structure of the ground-state complex formed in the presence of the GTP analog GMPPCP. However, small shifts that do occur within the shared catalytic chamber may be functionally important. Remarkably, an external nucleotide interaction site was identified at the activation region, revealed by an unexpected contaminating GMP molecule bound adjacent to the catalytic chamber. This site exhibits conserved sequence and structural features that suggest a direct interaction with RNA plays a role in regulating the activity of the SRP targeting complex.

Topics: Aluminum Compounds; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Dimerization; Fluorides; Fluorometry; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Magnesium; Models, Molecular; Molecular Conformation; Protein Binding; Receptors, Cytoplasmic and Nuclear; RNA, Bacterial; Signal Recognition Particle; Thermus

End binding 1 (EB1) proteins are highly conserved regulators of microtubule dynamics. Using electron microscopy (EM) and high-resolution surface shadowing we have studied the microtubule-binding properties of the fission yeast EB1 homolog Mal3p. This allowed for a direct visualization of Mal3p bound on the surface of microtubules. Mal3p particles usually formed a single line on each microtubule along just one of the multiple grooves that are formed by adjacent protofilaments. We provide structural data showing that the alignment of Mal3p molecules coincides with the microtubule lattice seam as well as data suggesting that Mal3p not only binds but also stabilizes this seam. Accordingly, Mal3p stabilizes microtubules through a specific interaction with what is potentially the weakest part of the microtubule in a way not previously demonstrated. Our findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with target structures such as microtubule-associated proteins, motors, kinetochores, or membranes.

Topics: Cytoskeleton; Guanosine Triphosphate; Microtubule Proteins; Microtubule-Associated Proteins; Models, Molecular; Models, Structural; Paclitaxel; Schizosaccharomyces pombe Proteins

The guanine nucleotide-binding protein Ras occurs in solution in two different states, state 1 and state 2, when the GTP analogue GppNHp is bound to the active center as detected by (31)P NMR spectroscopy. Here we show that Ras(wt).Mg(2+).GppCH(2)p also exists in two conformational states in dynamic equilibrium. The activation enthalpy DeltaH(++)(12) and the activation entropy DeltaS(++)(12) for the transition from state 1 to state 2 are 70 kJ mol(-1) and 102 J mol(-1) K(-1), within the limits of error identical to those determined for the Ras(wt).Mg(2+).GppNHp complex. The same is true for the equilibrium constants K(12) = [2]/[1] of 2.0 and the corresponding DeltaG(12) of -1.7 kJ mol(-1) at 278 K. This excludes a suggested specific effect of the NH group of GppNHp on the equilibrium. The assignment of the phosphorus resonance lines of the bound analogues has been done by two-dimensional (31)P-(31)P NOESY experiments which lead to a correction of the already reported assignments of bound GppNHp. Mutation of Thr35 in Ras.Mg(2+).GppCH(2)p to serine leads to a shift of the conformational equilibrium toward state 1. Interaction of the Ras binding domain (RBD) of Raf kinase or RalGDS with Ras(wt) or Ras(T35S) shifts the equilibrium completely to state 2. The (31)P NMR experiments suggest that, besides the type of the side chain of residue 35, a main contribution to the conformational equilibrium in Ras complexes with GTP and GTP analogues is the effective acidity of the gamma-phosphate group of the bound nucleotide. A reaction scheme for the Ras-effector interaction is presented which includes the existence of two conformations of the effector loop and a weak binding state.

Topics: Amino Acid Substitution; Cations, Divalent; Deuterium Exchange Measurement; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Kinetics; Magnesium; Magnetic Resonance Spectroscopy; Phosphates; Phosphorus Isotopes; Protein Binding; Protein Conformation; Proto-Oncogene Proteins c-raf; ral Guanine Nucleotide Exchange Factor; ras Proteins; Thermodynamics

FtsZ, the prokaryotic homologue of tubulin, is an essential cell division protein. In the cell, it localizes at the center, forming a ring that constricts during division. In vitro, it binds and hydrolyzes GTP and polymerizes in a GTP-dependent manner. We have used atomic force microscopy to study the structure and dynamics of FtsZ polymer assembly on a mica surface under buffer solution. The polymers were highly dynamic and flexible, and they continuously rearranged over the surface. End-to-end joining of filaments and depolymerization from internal zones were observed, suggesting that fragmentation and reannealing may contribute significantly to the dynamics of FtsZ assembly. The shape evolution of the restructured polymers manifested a strong inherent tendency to curve. Polymers formed in the presence of non-hydrolyzable nucleotide analogues or in the presence of GDP and AlF(3) were structurally similar but showed a slower dynamic behavior. These results provide experimental evidence supporting the model of single-strand polymerization plus cyclization recently proposed to explain the hydrodynamic behavior of the polymers in solution.

Topics: Adsorption; Aluminum Compounds; Aluminum Silicates; Chemical Phenomena; Chemistry, Physical; Escherichia coli; Escherichia coli Proteins; Fluorides; Guanosine Diphosphate; Guanosine Triphosphate; Hydrogen-Ion Concentration; Microscopy, Atomic Force; Polymers; Solutions

Topics: Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Microtubules; Pliability; Protein Conformation; Tubulin

The atomic structure of tubulin in a polymerized, straight protofilament is clearly distinct from that in a curved conformation bound to a cellular depolymerizer. The nucleotide contents are identical, and in both cases the conformation of the GTP-containing, intra-dimer interface is indistinguishable from the GDP-containing, inter-dimer contact. Here we present two structures corresponding to the start and end points in the microtubule polymerization and hydrolysis cycles that illustrate the consequences of nucleotide state on longitudinal and lateral assembly. In the absence of depolymerizers, GDP-bound tubulin shows distinctive intra-dimer and inter-dimer interactions and thus distinguishes the GTP and GDP interfaces. A cold-stable tubulin polymer with the non-hydrolysable GTP analogue GMPCPP, containing semi-conserved lateral interactions, supports a model in which the straightening of longitudinal interfaces happens sequentially, starting with a conformational change after GTP binding that straightens the dimer enough for the formation of lateral contacts into a non-tubular intermediate. Closure into a microtubule does not require GTP hydrolysis.

Topics: Cryoelectron Microscopy; Dimerization; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Microtubules; Models, Molecular; Pliability; Protein Conformation; Tubulin

GTP cyclohydrolase II converts GTP to 2,5-diamino-6-beta-ribosyl-4(3H)-pyrimidinone 5'-phosphate, formate and pyrophosphate, the first step in riboflavin biosynthesis. The essential role of riboflavin in metabolism and the absence of GTP cyclohydrolase II in higher eukaryotes makes it a potential novel selective antimicrobial drug target. GTP cyclohydrolase II catalyzes a distinctive overall reaction from GTP cyclohydrolase I; the latter converts GTP to dihydroneopterin triphosphate, utilized in folate and tetrahydrobiopterin biosynthesis. The structure of GTP cyclohydrolase II determined at 1.54-A resolution reveals both a different protein fold to GTP cyclohydrolase I and distinctive molecular recognition determinants for GTP; although in both enzymes there is a bound catalytic zinc. The GTP cyclohydrolase II.GMPCPP complex structure shows Arg(128) interacting with the alpha-phosphonate, and thus in the case of GTP, Arg(128) is positioned to act as the nucleophile for pyrophosphate release and formation of the proposed covalent guanylyl-GTP cyclohydrolase II intermediate. Tyr(105) is identified as playing a key role in GTP ring opening; it is hydrogen-bonded to the zinc-activated water molecule, the latter being positioned for nucleophilic attack on the guanine C-8 atom. Although GTP cyclohydrolase I and GTP cyclohydrolase II both use a zinc ion for the GTP ring opening and formate release, different residues are utilized in each case to catalyze this reaction step.

Topics: Binding Sites; Crystallization; Crystallography, X-Ray; Escherichia coli; GTP Cyclohydrolase; Guanosine Triphosphate; Molecular Structure; Protein Conformation; Tyrosine; Zinc

Microtubule assembly and disassembly is a complex structural process that does not proceed by simple addition and subtraction of individual subunits to and from a helical polymer, as would be the case for actin and other helical assemblies. The dynamic process of microtubule growth and shrinking involves short-lasting polymer forms that differ substantially from the microtubule itself and constitute crucial assembly and disassembly intermediates. Structural characterization thus depends on the stabilization of these brief intermediates and their preservation as polymeric assemblies. This paper gives experimental details on the polymerization of GMPCPP-tubulin into low-temperature, stable polymers that we propose to correspond to the early stages in microtubule assembly, and includes new data on the effect of colchicine on GMPCPP-tubulin polymerization. Finally, we add our thoughts on the possible biological meaning of tubulin polymerization versatility.

Topics: Animals; Colchicine; Cryoelectron Microscopy; Gout Suppressants; Guanosine Diphosphate; Guanosine Triphosphate; Image Processing, Computer-Assisted; Microtubule-Associated Proteins; Microtubules; Models, Biological; Polymers; Protein Binding; Protein Structure, Secondary; Temperature; Tubulin

The mitotic checkpoint is the major cell cycle control mechanism for maintaining chromosome content in multicellular organisms. Prevention of premature onset of anaphase requires activation at unattached kinetochores of the BubR1 kinase, which acts with other components to generate a diffusible "stop anaphase" inhibitor. Not only does direct binding of BubR1 to the centromere-associated kinesin family member CENP-E activate its essential kinase, binding of a motorless fragment of CENP-E is shown here to constitutively activate BubR1 bound at kinetochores, producing checkpoint signaling that is not silenced either by spindle microtubule capture or the tension developed at those kinetochores by other components. Using purified BubR1, microtubules, and CENP-E, microtubule capture by the CENP-E motor domain is shown to silence BubR1 kinase activity in a ternary complex of BubR1-CENP-E-microtubule. Together, this reveals that CENP-E is the signal transducing linker responsible for silencing BubR1-dependent mitotic checkpoint signaling through its capture at kinetochores of spindle microtubules.

Topics: Animals; Cattle; Cell Cycle Proteins; Cell Extracts; Cell Line, Tumor; Chromosomal Proteins, Non-Histone; Enzyme Activation; Female; Fluorescent Antibody Technique; Fluorescent Dyes; Gene Silencing; Glutathione Transferase; Guanosine Triphosphate; Humans; Kinetochores; Microtubules; Mitosis; Models, Biological; Oocytes; Point Mutation; Protein Kinases; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Recombinant Proteins; Rhodamines; Signal Transduction; Tubulin; Xenopus

Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. The structure explains the coordinate activation of the two GTPases. Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon. We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.

Topics: Amino Acid Motifs; Bacterial Proteins; Binding Sites; Catalysis; Crystallography, X-Ray; Dimerization; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits; Receptors, Cytoplasmic and Nuclear; Signal Recognition Particle; Thermus

Cycling between a GTP bound "on" state and a GDP bound "off" state, guanine nucleotide-binding (GNB) proteins act as molecular switches. The switching process and the interaction with effectors, GTPase-activating proteins, and guanosine nucleotide-exchange factors is accompanied by pronounced conformational changes of the switch regions of the GNB proteins. The aim of the present contribution is to correlate conformational changes observed by liquid-state NMR with solid-state (31)P NMR data and with the results of X-ray crystallography. Crystalline wild-type Ras complexed with GTP analogs such as GppCH(2)p and GppNHp could be prepared. At low temperatures, two different signals were found for the gamma-phosphate group of GppNHp bound to wild-type Ras. This behavior indicates the existence of two different conformations of the molecule in the crystalline state as it is found in solution but not by X-ray crystallography. In contrast to the GppNHp complex, the two separate gamma-phosphate signals could not be observed for GppCH(2)p bound to wild-type Ras. However, an increasing linewidth at low temperature indicates the presence of an exchange process. The results obtained for the wild-type protein are compared with the behavior of GppNHp complexes of the effector loop mutants Ras(T35S) and Ras(T35A). These mutants prefer a conformation similar to the GDP bound "off" state.

Topics: Binding Sites; Crystallization; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; In Vitro Techniques; Models, Molecular; Mutagenesis, Site-Directed; Nuclear Magnetic Resonance, Biomolecular; Phosphorus; Protein Conformation; ras Proteins; Recombinant Proteins; Solutions

Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus egg extracts. This depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI). We purified the factor responsible for this novel depolymerizing activity using biochemical fractionation and a visual activity assay and identified it as XMAP215, previously identified as a prominent MT growth-promoting protein in Xenopus extracts. Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro. Stimulation of depolymerization is specific for the MT plus end. These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions. We propose that the substrate in our assay, GMPCPP-stabilized MTs, serves as a model for the pause state of MT ends and that the multiple activities of XMAP215 are unified by a mechanism of antagonizing MT pauses.

Topics: Animals; Biological Assay; Cell Extracts; Female; Guanosine Triphosphate; Microtubule Proteins; Microtubule-Associated Proteins; Microtubules; Models, Biological; Oocytes; Protein Structure, Tertiary; Recombinant Fusion Proteins; Xenopus laevis; Xenopus Proteins

Mobility of taxol inside microtubules was investigated using fluorescence recovery after photobleaching on flow-aligned bundles. Bundles were made of microtubules with either GMPCPP or GTP at the exchangeable site on the tubulin dimer. Recovery times were sensitive to bundle thickness and packing, indicating that taxol molecules are able to move laterally through the bundle. The density of open binding sites along a microtubule was varied by controlling the concentration of taxol in solution for GMPCPP samples. With >63% sites occupied, recovery times were independent of taxol concentration and, therefore, inversely proportional to the microscopic dissociation rate, k(off). It was found that 10k(off)(GMPCPP) approximately equal k(off)(GTP), consistent with, but not fully accounting for, the difference in equilibrium constants for taxol on GMPCPP and GTP microtubules. With <63% sites occupied, recovery times decreased as approximately [Tax](-1/5) for both types of microtubules. We conclude that the diffusion of taxol inside the microtubule bundle is hindered by rebinding events when open sites are within approximately 7 nm of each other.

Topics: Binding Sites; Biomimetic Materials; Diffusion; Dimerization; Fluorescence Recovery After Photobleaching; Guanosine Triphosphate; Membranes, Artificial; Microtubules; Motion; Paclitaxel; Protein Binding; Tubulin

The GTPases Ffh and FtsY are components of the prokaryotic signal recognition particle protein-targeting pathway. The two proteins interact in a GTP-dependent manner, forming a complex that can be stabilized by use of the non-hydrolyzable GTP analog GMPPCP. Crystals of the complex of the NG GTPase domains of the two proteins have been obtained from ammonium sulfate solutions. Crystals grow with several different morphologies, predominately as poorly diffracting plates and needle clusters, but occasionally as well diffracting rods. It has been demonstrated that all forms of the crystals observed contain an intact complex. Diffraction data to 2.0 A resolution have been measured.

Topics: Bacterial Proteins; Crystallization; Crystallography, X-Ray; Escherichia coli Proteins; GTP Phosphohydrolases; Guanosine Triphosphate; Protein Structure, Tertiary; Receptors, Cytoplasmic and Nuclear; Signal Recognition Particle; Thermus

Cdc42Hs is a signal transduction protein that is involved in cytoskeletal growth and organization. We describe here the methyl side chain dynamics of three forms of (2)H,(13)C,(15)N-Cdc42Hs [GDP-bound (inactive), GMPPCP-bound (active), and GMPPCP/PBD46-bound (effector-bound)] from (13)C-(1)H NMR measurements of deuterium T(1) and T(1 rho) relaxation times. A wide variation in flexibility was observed throughout the protein, with methyl axis order parameters (S(2)(axis)) ranging from 0.2 to 0.4 (highly disordered) in regions near the PBD46 binding site to 0.8--1.0 (highly ordered) in some helices. The side chain dynamics of the GDP and GMPPCP forms are similar, with methyl groups on the PBD46 binding surface experiencing significantly greater mobility (lower S(2)(axis)) than those not on the binding surface. Binding of PBD46 results in a significant increase in the disorder and a corresponding increase in entropy for the majority of methyl groups. Many of the methyl groups that experience an increase in mobility are found in residues that are not part of the PBD46 binding interface. This entropy gain represents a favorable contribution to the overall entropy of effector binding and partially offsets unfavorable entropy losses such as those that occur in the backbone.

Topics: Amino Acid Sequence; Animals; cdc42 GTP-Binding Protein; Deuterium; Entropy; Enzyme Activation; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Ligands; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; p21-Activated Kinases; Peptide Fragments; Protein Binding; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Solutions; Thermodynamics

When isolated from tissues, the alpha beta-dimeric protein tubulin consists of multiple isoforms which originate from the expression and subsequent posttranslational modification of multiple polypeptide sequences. Microtubules studied in vitro consist of mixtures of these isoforms. It is therefore not known whether dimers composed of single sequences of alpha- and beta-tubulin can polymerize to form microtubules, or whether posttranslational modifications may be necessary for microtubule assembly. To initiate investigation of these questions, rabbit reticulocyte lysate, which contains the cytoplasmic chaperonin CCT and its cofactors, was employed to prepare substantial quantities (tens of micrograms) of active tubulin by in vitro folding of mouse alpha- and beta-tubulins recombinantly synthesized in E. coli. This recombinant tubulin is composed of only a single alpha-chain and a single beta-chain. When analyzed after folding by isoelectric focusing, each chain yielded only one band, indicating that neither was detectably posttranslationally modified in the course of the folding reaction. When subjected to assembly-promoting conditions, this tubulin formed microtubules without the addition of any exogenous protein. Electron microscopy showed them to be of normal morphology. Analysis of their protein composition showed that they are composed nearly entirely of recombinant tubulin. These results demonstrate that the naturally occurring mixtures of isoforms are not strictly required for the formation of microtubules. They also open a route to other studies, both biomedical and structural, of fully defined tubulin in vitro.

Topics: Animals; Cattle; Dimerization; Escherichia coli; Genetic Vectors; Guanosine Triphosphate; Isoelectric Focusing; Mice; Microtubules; Muscle Proteins; Protein Folding; Rabbits; Recombinant Proteins; Solutions; Tubulin

Microtubule architecture can vary with eukaryotic species, with different cell types, and with the presence of stabilizing agents. For in vitro assembled microtubules, the average number of protofilaments is reduced by the presence of sarcodictyin A, epothilone B, and eleutherobin (similarly to taxol) but increased by taxotere. Assembly with a slowly hydrolyzable GTP analogue GMPCPP is known to give 96% 14 protofilament microtubules. We have used electron cryomicroscopy and helical reconstruction techniques to obtain three-dimensional maps of taxotere and GMPCPP microtubules incorporating data to 14 A resolution. The dimer packing within the microtubule wall is examined by docking the tubulin crystal structure into these improved microtubule maps. The docked tubulin and simulated images calculated from "atomic resolution" microtubule models show tubulin heterodimers are aligned head to tail along the protofilaments with the beta subunit capping the microtubule plus end. The relative positions of tubulin dimers in neighboring protofilaments are the same for both types of microtubule, confirming that conserved lateral interactions between tubulin subunits are responsible for the surface lattice accommodation observed for different microtubule architectures. Microtubules with unconventional protofilament numbers that exist in vivo are likely to have the same surface lattice organizations found in vitro. A curved "GDP" tubulin conformation induced by stathmin-like proteins appears to weaken lateral contacts between tubulin subunits and could block microtubule assembly or favor disassembly. We conclude that lateral contacts between tubulin subunits in neighboring protofilaments have a decisive role for microtubule stability, rigidity, and architecture.

Topics: Actin Cytoskeleton; Alkaloids; Amino Acid Sequence; Animals; Dimerization; Diterpenes; Docetaxel; Epothilones; Epoxy Compounds; Excipients; Guanosine Triphosphate; Microtubules; Molecular Sequence Data; Paclitaxel; Protein Conformation; Surface Properties; Swine; Taxoids; Thiazoles; Tubulin

Cdc42Hs is a member of the Ras superfamily of GTPases which, when active, initiates a cascade beginning with the activation of several kinases, including P(21)-activated kinase (PAK). We previously determined the structure of a complex between a 46 amino acid fragment peptide derived from the PAK binding domain (PBD46) and Cdc42Hs.GMPPCP (Gizachew, D., Guo, W., Chohan, K. K., Sutcliffe, M. J., and Oswald, R. E. (2000) Biochemistry 39, 3963-3971). Previous studies (Loh, A. P., Guo, W., Nicholson, L. K., and Oswald, R. E. (1999) Biochemistry 38, 12547-12557) suggest that the regions of Cdc42Hs that bind effectors and regulators have distinct dynamic properties from the remainder of the protein. Here, we describe the backbone dynamics of PBD46 bound to Cdc42Hs.GMPPCP. T(1), T(2), T(1)(rho), and steady-state nuclear Overhauser effects were measured at 500 and 600 MHz. An extension of the Lipari-Szabo model-free analysis was used to determine the order parameters (S(2)) and local correlation times (tau(e)) of the N-H bond vectors within PBD46. Both Cdc42Hs and PBD46 exhibit increased mobility in the free versus the bound state, suggesting that protein flexibility may be required for high-affinity PBD46 binding and, presumably, the activation of PAK. Different backbone dynamics were observed in different regions of the peptide. The beta-strand region of bound PBD46, which makes contacts with beta2 of Cdc42Hs, exhibits low mobility on the pico- to nanosecond timescale. However, the part of PBD46 that interacts with Switch I of Cdc42Hs exhibits greater mobility. Thus, PBD46 and Cdc42Hs form a tight complex that exhibits concerted dynamics.

Topics: Amino Acid Sequence; Guanosine Triphosphate; Models, Molecular; Molecular Sequence Data; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; p21-Activated Kinases; Peptide Fragments; Protein Binding; Protein Conformation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proteins; Recombinant Proteins; Wiskott-Aldrich Syndrome Protein

FtsZ assembles in vitro into protofilaments that can adopt two conformations-the straight conformation, which can assemble further into two-dimensional protofilament sheets, and the curved conformation, which forms minirings about 23 nm in diameter. Here, we describe the structure of FtsZ tubes, which are a variation of the curved conformation. In the tube the curved protofilament forms a shallow helix with a diameter of 23 nm and a pitch of 18 or 24 degrees. We suggest that this shallow helix is the relaxed structure of the curved protofilament in solution. We provide evidence that GTP favors the straight conformation while GDP favors the curved conformation. In particular, exclusively straight protofilaments and protofilament sheets are assembled in GMPCPP, a nonhydrolyzable GTP analog, or in GTP following chelation of Mg, which blocks GTP hydrolysis. Assembly in GDP produces exclusively tubes. The transition from straight protofilaments to the curved conformation may provide a mechanism whereby the energy of GTP hydrolysis is used to generate force for the constriction of the FtsZ ring in cell division.

Topics: Actin Cytoskeleton; Bacterial Proteins; Calcium; Chelating Agents; Cytoskeletal Proteins; DEAE-Dextran; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Magnesium; Microscopy, Electron; Protein Conformation

A fluorescent derivative of paclitaxel, 3'-N-m-aminobenzamido-3'-N-debenzamidopaclitaxel (N-AB-PT), has been prepared in order to probe paclitaxel-microtubule interactions. Fluorescence spectroscopy was used to quantitatively assess the association of N-AB-PT with microtubules. N-AB-PT was found equipotent with paclitaxel in promoting microtubule polymerization. Paclitaxel and N-AB-PT underwent rapid exchange with each other on microtubules assembled from GTP-, GDP-, and GMPCPP-tubulin. The equilibrium binding parameters for N-AB-PT to microtubules assembled from GTP-tubulin were derived through fluorescence titration. N-AB-PT bound to two types of sites on microtubules (K(d1) = 61 +/- 7.0 nM and K(d2) = 3.3 +/- 0.54 microM). The stoichiometry of each site was less than one ligand per tubulin dimer in the microtubule (n(1) = 0.81 +/- 0.03 and n(2) = 0.44 +/- 0.02). The binding experiments were repeated after exchanging the GTP for GDP or for GMPCPP. It was found that N-AB-PT bound to a single site on microtubules assembled from GDP-tubulin with a dissociation constant of 2.5 +/- 0.29 microM, and that N-AB-PT bound to a single site on microtubules assembled from GMPCPP-tubulin with a dissociation constant of 15 +/- 4.0 nM. It therefore appears that microtubules contain two types of binding sites for paclitaxel and that the binding site affinity for paclitaxel depends on the nucleotide content of tubulin. It has been established that paclitaxel binding does not inhibit GTP hydrolysis and microtubules assembled from GTP-tubulin in the presence of paclitaxel contain almost exclusively GDP at the E-site. We propose that although all the subunits of the microtubule at steady state are the same "GDP-tubulin-paclitaxel", they are formed through two paths: paclitaxel binding to a tubulin subunit before its E-site GTP hydrolysis is of high affinity, and paclitaxel binding to a tubulin subunit containing hydrolyzed GDP at its E-site is of low affinity.

Topics: Animals; Brain; Cattle; Fluorescent Dyes; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Microtubules; Paclitaxel; Taxoids; Tubulin

The ribosome undergoes pronounced periodic conformational changes during protein synthesis. Of particular importance are those occurring around the decoding site, the region of the 16 S rRNA interacting with the mRNA-(tRNA)(2) complex. We have incorporated structural information from X-ray crystallography and nuclear magnetic resonance into cryo-electron microscopic maps of ribosomal complexes designed to capture structural changes at the translocation step of the polypeptide elongation cycle. The A-site region of the decoding site actively participates in the translocation of the tRNA from the A to the P-site upon GTP hydrolysis by elongation factor G, shifting approximately 8 A toward the P-site. This implies that elongation factor G actively pushes both the decoding site and the mRNA/tRNA complex during translocation.

Topics: Base Sequence; Binding Sites; Cryoelectron Microscopy; Crystallography, X-Ray; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acid Conformation; Peptide Chain Elongation, Translational; Peptide Elongation Factor G; Protein Conformation; Ribosomes; RNA, Ribosomal, 16S; RNA, Transfer

Bacterial cell division protein FtsZ assembles into protofilaments, which can adopt a straight or curved conformation, similar to its eukaryotic homolog, tubulin. The straight protofilaments can assemble into sheets with a lattice similar to the microtubule wall. The curved protofilaments can form rings when adsorbed to a lipid monolayer, but in solution they form helices. 4 helices assemble together to make a tube, the characteristic polymer of the curved protofilament. GTP favors the straight conformation, while GDP favors the curved. We show here that addition of EDTA and GTP to tubes causes a rapid transformation to straight protofilament sheets. Apparently when the magnesium is chelated the GDP in the curved protofilaments dissociates rapidly and is replaced with GTP, and this GTP induces the transition to straight protofilaments.

Topics: Bacterial Proteins; Calcium; Cytoskeletal Proteins; DEAE-Dextran; Edetic Acid; Guanosine Diphosphate; Guanosine Triphosphate; Lipid Bilayers; Macromolecular Substances; Magnesium; Microscopy, Electron; Models, Molecular

The molecular mechanisms that allow elongation of the unstable microtubule lattice remain unclear. It is usually thought that the GDP-liganded tubulin lattice is capped by a small layer of GTP- or GDP-Pi-liganded molecules, the so called "GTP-cap". Here, we point-out that the elastic properties of the microtubule lattice cause a difference in stability between the elongating tubulin sheet and the completed microtubule wall. The implications of our observations for microtubule structure and dynamics are discussed.

Topics: Binding Sites; Computer Simulation; Cryoelectron Microscopy; Elasticity; Guanosine Diphosphate; Guanosine Triphosphate; Macromolecular Substances; Microtubules; Models, Biological; Models, Molecular; Protein Binding; Protein Conformation; Thermodynamics; Tubulin

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein that destabilizes microtubules, but the mechanism of destabilization is currently controversial. Based on in vitro microtubule assembly assays, evidence has been presented supporting conflicting destabilization models of either tubulin sequestration or promotion of microtubule catastrophes. We found that Op18 can destabilize microtubules by both of these mechanisms and that these activities can be dissociated by changing pH. At pH 6.8, Op18 slowed microtubule elongation and increased catastrophes at both plus and minus ends, consistent with a tubulin-sequestering activity. In contrast, at pH 7.5, Op18 promoted microtubule catastrophes, particularly at plus ends, with little effect on elongation rates at either microtubule end. Dissociation of tubulin-sequestering and catastrophe-promoting activities of Op18 was further demonstrated by analysis of truncated Op18 derivatives. Lack of a C-terminal region of Op18 (aa 100-147) resulted in a truncated protein that lost sequestering activity at pH 6.8 but retained catastrophe-promoting activity. In contrast, lack of an N-terminal region of Op18 (aa 5-25) resulted in a truncated protein that still sequestered tubulin at pH 6.8 but was unable to promote catastrophes at pH 7.5. At pH 6. 8, both the full length and the N-terminal-truncated Op18 bound tubulin, whereas truncation at the C-terminus resulted in a pronounced decrease in tubulin binding. Based on these results, and a previous study documenting a pH-dependent change in binding affinity between Op18 and tubulin, it is likely that tubulin sequestering observed at lower pH resulted from the relatively tight interaction between Op18 and tubulin and that this tight binding requires the C-terminus of Op18; however, under conditions in which Op18 binds weakly to tubulin (pH 7.5), Op18 stimulated catastrophes without altering tubulin subunit association or dissociation rates, and Op18 did not depolymerize microtubules capped with guanylyl (alpha, beta)-methylene diphosphonate-tubulin subunits. We hypothesize that weak binding between Op18 and tubulin results in free Op18, which is available to interact with microtubule ends and thereby promote catastrophes by a mechanism that likely involves GTP hydrolysis.

Topics: Animals; Binding Sites; Cattle; Dimerization; DNA Primers; Drug Stability; Guanosine Triphosphate; Hydrogen-Ion Concentration; In Vitro Techniques; Microtubule Proteins; Microtubules; Peptide Fragments; Phosphoproteins; Protein Binding; Protein Conformation; Sea Urchins; Sequence Deletion; Stathmin; Swine; Tubulin

CLIP-170 is a cytoplasmic linker protein that localizes to plus ends of microtubules in vivo. In this study, we have characterized the microtubule-binding properties of CLIP-170, to understand the mechanism of its plus end targeting. We show that the NH2-terminal microtubule-interacting domain of CLIP-170 alone localizes to microtubule plus ends when transfected into cells. Association of CLIP-170 with newly-formed microtubules was observed in cells microinjected with biotinylated tubulin, used as a tracer for growing microtubules. Using in vitro assays, association of CLIP-170 with recently polymerized tubulin is also seen. Cross-linking and sedimentation velocity experiments suggest association of CLIP-170 with nonpolymerized tubulin. We conclude from these experiments that the microtubule end targeting of CLIP-170 is closely linked to tubulin polymerization.

Topics: Centrosome; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; HeLa Cells; Humans; Microtubule-Associated Proteins; Microtubules; Neoplasm Proteins; Tubulin; Tumor Cells, Cultured

Tissue transglutaminase (tTG) catalyzes a Ca(2+)-dependent transglutaminase (TGase) activity that stabilizes tissues and a GTP hydrolysis activity that regulates cell receptor signaling. The purpose of this study was to examine the true substrates for nucleotide hydrolysis and the effects of these substrates on modulating the dual enzymatic activities of tTG. We found that Mg-GTP and Mg-ATP are the true substrates of the hydrolysis reaction. tTG hydrolyzed Mg-GTP and Mg-ATP at similar rates and interacted with Mg-ATP (Km = 38 +/- 10 microM) at a 3-fold greater steady-state affinity than with Mg-GTP (Km = 130 +/- 35 microM). In addition, Mg-ATP inhibited GTP hydrolysis (IC50 = 24 microM), whereas 1 mM Mg-GTP reduced ATP hydrolysis by only 20%. Furthermore, the TGase activity of tTG was inhibited by Mg-GTP, Mg-GDP, and Mg-GMP, with IC50 values of 9, 9, and 400 microM, respectively, whereas the Mg-adenine nucleotides were ineffective. Kinetic analysis of the hydrolysis reaction demonstrates the presence of separate binding sites for Mg-GTP and Mg-ATP. Finally, we found that Mg-GTP protected tTG from proteolytic degradation by trypsin, whereas Mg-ATP was ineffective. In conclusion, we report that Mg-GTP and Mg-ATP can bind to distinct sites and serve as substrates for nucleotide hydrolysis. Furthermore, binding of Mg-GTP causes a conformational change and the inhibition of TGase activity, whereas Mg-ATP is ineffective. The implication of these findings in regulating the intracellular and extracellular function of tTG is discussed.

Topics: Adenosine Triphosphate; Calcium; Guanosine Triphosphate; Humans; Hydrolysis; In Vitro Techniques; Kinetics; Macromolecular Substances; Magnesium; Nucleotides; Protein Conformation; Transglutaminases

Microtubules are dynamic polymers that interconvert between periods of slow growth and fast shrinkage. The energy driving this nonequilibrium behavior comes from the hydrolysis of GTP, which is required to destabilize the microtubule lattice. To understand the mechanism of this destabilization, cryo-electron microscopy was used to compare the structure of the ends of shrinking microtubules assembled in the presence of either GTP or the slowly hydrolyzable analogue guanylyl (alpha,beta)methylenediphosphonate (GMPCPP). Depolymerization was induced by cold or addition of calcium. With either nucleotide, we have observed curled oligomers at the ends of shrinking microtubules. However, GDP oligomers were consistently more curved than GMPCPP oligomers. This difference in curvature between depolymerizing GDP and GMPCPP protofilaments suggests that GTP hydrolysis is accompanied by an increase in curvature of the protofilaments, thereby destabilizing the lateral interactions between tubulin subunits in the microtubule lattice.

Topics: Animals; Cattle; Dimerization; Guanosine Triphosphate; Hydrolysis; Microtubules

The Ras superfamily of GTP-binding proteins is involved in a number of cellular signaling events including, but not limited to, tumorigenesis, intracellular trafficking, and cytoskeletal organization. The Rho subfamily, of which Cdc42Hs is a member, is involved in cell morphogenesis through a GTPase cascade which regulates cytoskeletal changes. Cdc42Hs has been shown to stimulate DNA synthesis as well as to initiate a protein kinase cascade that begins with the activation of the p21-activated serine/threonine kinases (PAKs). We have determined previously the solution structure of Cdc42Hs [Feltham et al. (1997) Biochemistry 36, 8755-8766] using NMR spectroscopy. A minimal-binding domain of 46 amino acids of PAK was identified (PBD46), which binds Cdc42Hs with a KD of approximately 20 nM and inhibits GTP hydrolysis. The binding interface was mapped by producing a fully deuterated sample of 15N-Cdc42Hs bound to PBD46. A 1H,15N-NOESY-HSQC spectrum demonstrated that the binding surface on Cdc42Hs consists of the second beta-strand (beta2) and a portion of the loop between the first alpha-helix (alpha1) and beta2 (switch I). A complex of PBD46 bound to 15N-Cdc42Hs.GMPPCP exhibited extensive chemical shift changes in the 1H,15N-HSQC spectrum. Thus, PBD46 likely produces structural changes in Cdc42Hs which are not limited to the binding interface, consistent with its effects on GTP hydrolysis. These results suggest that the kinase-binding domain on Cdc42Hs is similar to, but more extensive than, the c-Raf-binding domain on the Ras antagonist, Rap1 [Nassar et al. (1995) Nature 375, 554-560)].

Topics: Amino Acid Sequence; Binding Sites; cdc42 GTP-Binding Protein; Cell Cycle Proteins; Escherichia coli; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; p21-Activated Kinases; Protein Binding; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Recombinant Proteins

Different models have been proposed that link the tubulin heterodimer nucleotide content and the role of GTP hydrolysis with microtubule assembly and dynamics. Here we compare the thermodynamics of microtubule assembly as a function of nucleotide content by van't Hoff analysis. The thermodynamic parameters of tubulin assembly in 30-100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM MgSO4, 2 mM EGTA, pH 6.9, in the presence of a weakly hydrolyzable analog, GMPCPP, the dinucleotide analog GMPCP plus 2 M glycerol, and GTP plus 2 M glycerol were obtained together with data for taxol-GTP/GDP tubulin assembly (GMPCPP and GMPCP are the GTP and GDP nucleotide analogs where the alpha beta oxygen has been replaced by a methylene, -CH2-). All of the processes studied are characterized by a positive enthalpy, a positive entropy, and a large, negative heat capacity change. GMPCP-induced assembly has the largest negative heat capacity change and GMPCPP has the second largest, whereas GTP/2 M glycerol- and taxol-induced assembly have more positive values, respectively. A large, negative heat capacity is most consistent with the burial of water-accessible hydrophobic surface area, which gives rise to the release of bound water. The heat capacity changes observed with GTP/2 M glycerol-induced and with taxol-induced assembly are very similar, -790 +/- 190 cal/mol/k, and correspond to the burial of 3330 +/- 820 A2 of nonpolar surface area. This value is shown to be very similar to an estimate of the buried nonpolar surface in a reconstructed microtubule lattice. Polymerization data from GMPCP- and GMPCPP-induced assembly are consistent with buried nonpolar surface areas that are 3 and 6 times larger. A linear enthalpy-entropy and enthalpy-free energy plot for tubulin polymerization reactions verifies that enthalpy-entropy compensation for this system is based upon true biochemical correlation, most likely corresponding to a dominant hydrophobic effect. Entropy analysis suggests that assembly with GTP/2 M glycerol and with taxol is consistent with conformational rearrangements in 3-6% of the total amino acids in the heterodimer. In addition, taxol binding contributes to the thermodynamics of the overall process by reducing the delta H degree and delta S degree for microtubule assembly. In the presence of GMPCPP or GMPCP, tubulin subunits associate with extensive conformational rearrangement, corresponding to 10% and 26% of the total amino acids in the heterodimer, respe

Topics: Animals; Colchicine; Computer Simulation; Dimerization; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Indicators and Reagents; Microscopy, Electron; Microtubules; Monte Carlo Method; Nephelometry and Turbidimetry; Paclitaxel; Protein Conformation; Swine; Thermodynamics; Tubulin

The current two-state GTP cap model of microtubule dynamic instability proposes that a terminal crown of GTP-tubulin stabilizes the microtubule lattice and promotes elongation while loss of this GTP-tubulin cap converts the microtubule end to shortening. However, when this model was directly tested by using a UV microbeam to sever axoneme-nucleated microtubules and thereby remove the microtubule's GTP cap, severed plus ends rapidly shortened, but severed minus ends immediately resumed elongation (Walker, R.A., S. Inoué, and E.D. Salmon. 1989. J. Cell Biol. 108: 931-937). To determine if these previous results were dependent on the use of axonemes as seeds or were due to UV damage, or if they instead indicate an intermediate state in cap dynamics, we performed UV cutting of self-assembled microtubules and mechanical cutting of axoneme-nucleated microtubules. These independent methods yielded results consistent with the original work: a significant percentage of severed minus ends are stable after cutting. In additional experiments, we found that the stability of both severed plus and minus ends could be increased by increasing the free tubulin concentration, the solution GTP concentration, or by assembling microtubules with guanylyl-(alpha,beta)-methylene-diphosphonate (GMPCPP). Our results show that stability of severed ends, particularly minus ends, is not an artifact, but instead reveals the existence of a metastable kinetic intermediate state between the elongation and shortening states of dynamic instability. The kinetic properties of this intermediate state differ between plus and minus ends. We propose a three-state conformational cap model of dynamic instability, which has three structural states and four transition rate constants, and which uses the asymmetry of the tubulin heterodimer to explain many of the differences in dynamic instability at plus and minus ends.

Topics: Animals; Electrochemistry; Guanosine Triphosphate; Magnesium; Microtubules; Solutions; Swine; Tubulin; Ultraviolet Rays

It is well established that microtubules interact with intracellular membranes of eukaryotic cells. There is also evidence that tubulin, the major subunit of microtubules, associates directly with membranes. In many cases, this association between tubulin and membranes involves hydrophobic interactions. However, neither primary sequence nor known posttranslational modifications of tubulin can account for such an interaction. The goal of this study was to determine the molecular nature of hydrophobic interactions between tubulin and membranes. Specifically, I sought to identify a posttranslational modification of tubulin that is found in membrane proteins but not in cytoplasmic proteins. One such modification is the covalent attachment of the long chain fatty acid palmitate. The possibility that tubulin is a substrate for palmitoylation was investigated. First, I found that tubulin was palmitoylated in resting platelets and that the level of palmitoylation of tubulin decreased upon activation of platelets with thrombin. Second, to obtain quantities of palmitoylated tubulin required for protein structure analysis, a cell-free system for palmitoylation of tubulin was developed and characterized. The substrates for palmitoylation were nonpolymerized tubulin and tubulin in microtubules assembled with the slowly hydrolyzable GTP analogue guanylyl-(alpha, beta)-methylene-diphosphonate. However, tubulin in Taxol-assembled microtubules was not a substrate for palmitoylation. Likewise, palmitoylation of tubulin in the cell-free system was specifically inhibited by the antimicrotubule drugs Colcemid, podophyllotoxin, nocodazole, and vinblastine. These experiments identify a previously unknown posttranslational modification of tubulin that can account for at least one type of hydrophobic interaction with intracellular membranes.

Topics: Animals; Blood Platelets; Brain Chemistry; Cell Membrane; Cell-Free System; Demecolcine; Guanosine Triphosphate; Humans; Male; Microtubules; Nocodazole; Paclitaxel; Palmitoyl Coenzyme A; Podophyllotoxin; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Substrate Specificity; Swine; Thrombin; Tubulin; Vinblastine

It is now well established that guanine nucleotides are allosteric effectors of the vinca alkaloid-induced self-association of tubulin. GDP enhances self-association for vinblastine-, vincristine- and vinorelbine-induced spiral assembly relative to GTP by 0.90 +/- 0.17 kcal/mol [Lobert et al. (1996) Biochemistry 35, 6806-6814]. Since chemical modifications of the vinca alkaloid structure are known to modulate the overall affinity of drug binding, it is very likely that, by Wyman linkage, chemical modifications of guanine nucleotide allosteric effectors also modulate drug binding. Here we compare the effects of the GTP and GDP alpha,beta-methylene analogues GMPCPP and GMPCP on vinblastine-induced tubulin association in 10 and 100 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), 1 mM MgSO4, and 2 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), pH 6. 9, at different temperatures. We found that GMPCPP perfectly mimics GTP in its effect on spiral assembly under all ionic strength and temperature conditions. However, GMPCP in 10 mM Pipes behaves not as a GDP analogue, but as a GTP analogue. In 100 mM Pipes, GMPCP has characteristics that are intermediate between GDP and GTP. These data suggest that the alpha,beta methylene group in GMPCP and GMPCPP is sufficient to produce a GTP-like effect on vinblastine-induced tubulin self-assembly. This is consistent with previous observations that GMPCP-tubulin will assemble into microtubules in a 2 M glycerol and 100 mM Pipes buffer [Vulevic & Correia (1997) Biophys. J. 72, 1357-1375]. Our results demonstrate that an alpha,beta methylene modification of the guanine nucleotide phosphate moiety can induce a salt-dependent conformational change in the tubulin heterodimer that favors the GTP-tubulin structure. This has important implications for understanding allosteric interactions that occur in the binding of guanine nucleotides to tubulin.

Topics: Alkanesulfonic Acids; Animals; Brain; Calorimetry; Egtazic Acid; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Magnesium Sulfate; Piperazines; Swine; Tubulin; Vinblastine; Vincristine; Vinorelbine

The 5' cap of a mammalian pre-mRNA has been shown to interact with splicing components at the adjacent 5' splice site for processing of the first exon and the removal of the first intron (E. Izaurralde, J. Lewis, C. McGuigan, M. Jankowska, E. Darzynkiewicz, and I.W. Mattaj, Cell 78:657-668, 1994). Likewise, it has been shown that processing of the last exon and removal of the last intron involve interaction between splicing components at the 3' splice site and the polyadenylation complex at the polyadenylation signal (M. Niwa, S. D. Rose, and S.M. Berget, Genes Dev. 4:1552-1559, 1990; M. Niwa and S. M. Berget, Genes Dev. 5:2086-2095, 1991). These findings suggest that the cap provides a function in first exon processing which is similar to the function of the 3' splice site at last exon processing. To determine whether caps and 3' splice sites function similarly, we compared the effects of the cap and the 3' splice site on the in vitro utilization of the simian virus 40 late polyadenylation signal. We show that the presence of a m7GpppG cap, but not a cap analog, can positively affect the efficiency of polyadenylation of a polyadenylation-only substrate. Cap analogs do not stimulate polyadenylation because they fail to bind titratable cap-binding factors. The failure of cap analogs to stimulate polyadenylation can be overcome if a 3' splice site is present upstream of the polyadenylation signal. These data indicate that factors interacting with the cap or the 3' splice site function similarly to affect polyadenylation signal, along with m7GpppG cap, is inhibitory to polyadenylation. This finding suggests that the interaction between the cap-binding complexes and splicing components at the 5' splice site may form a complex which is inhibitory to further processing if splicing of an adjacent intron is not achieved.

Topics: Base Sequence; Binding Sites; Dinucleoside Phosphates; Exons; Guanosine Triphosphate; HeLa Cells; Humans; Introns; Molecular Structure; Poly A; RNA Caps; RNA Precursors; RNA Processing, Post-Transcriptional; RNA Splicing

The GTP analog guanylylmethylene diphosphonate (GppCH2p) strongly inhibited polyuridylic acid-directed polypeptide synthesis in a cell-free translation system prepared from Agrobacterium tumefaciens. Fusidic acid increased even further the inhibitory action. The pre-translocational ribosomal complexes formed with the GppCH2p and the elongation factor G protected the ribosome against the depurinating action of crotin 2 assayed as the acid-dependent release of the RNA fragment whose terminal sequence is 5'-GAGGACCGGGAUGGAC-3'. The results allowed to conclude that the interaction of both crotin 2 and the elongation factor G with the A. tumefaciens ribosomes in the pre-translocational state must take place at overlapping, either sterically or allosterically, ribosomal sites which are equally accessible to the RIP.

Topics: Agrobacterium tumefaciens; Base Sequence; Fusidic Acid; Guanosine Triphosphate; Molecular Sequence Data; Peptide Biosynthesis; Peptides; Plant Proteins; Poly U; Protein Biosynthesis; Protein Synthesis Inhibitors; Ribosome Inactivating Proteins, Type 1; Ribosomes; RNA, Bacterial; RNA, Ribosomal

Evidence that 13 or 14 contiguous tubulin-GTP subunits are sufficient to cap and stabilize a microtubule end and that loss of only one of these subunits results in the transition to rapid disassembly(catastrophe) was obtained using the slowly hydrolyzable GTP analogue guanylyl-(a,b)-methylene-diphosphonate (GMPCPP). The minus end of microtubules assembled with GTP was transiently stabilized against dilution-induced disassembly by reaction with tubulin-GMPCPP subunits for a time sufficient to cap the end with an average 40 subunits. The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was estimated from an observed 25- to 2000-s lifetime of the GMPCPP-stabilized microtubules following dilution with buffer and from the time required for loss of a single tubulin-GMPCPP subunit from the microtubule end (found to be 15 s). Rather than assuming that the 25- to 2000-s dispersion in cap lifetime results from an unlikely 80-fold range in the number of tubulin-GMPCpP subunits added in the 25-s incubation, it is proposed that this results because the minimum stable cap contains 13 to 14 tubulin-GMPCPP subunits. As a consequence, a microtubule capped with 13-14 tubulin-GMPCPP subunits switches to disassembly after only one dissociation event (in about 15 s), whereas the time required for catastrophe of a microtubule with only six times as many subunits (84 subunits) corresponds to 71 dissociation events (84-13). The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was also estimated with microtubules in which a GMPCPP-cap was formed by allowing chance to result in the accumulation of multiple contiguous tubulin-GMPCPP subunits at the end, during the disassembly of microtubules containing both GDP and GMPCPP. Our observation that the disassembly rate was inhibited in proportion to the 13-14th power of the fraction of subunits containing GMPCPP again suggests that a minimum cap contains 13-14 tubulin-GMPCPP subunits. A remeasurement of the rate constant for dissociation of a tubulin-GMPCPP subunit from the plus-end of GMPCPP microtubules, now found to be 0.118 s-1, has allowed a better estimate of the standard free energy for hydrolysis of GMPCPP in a microtubule and release of Pi: this is +0.7 kcal/mol, rather than -0.9 kcal/mol, as previously reported.

Topics: Animals; Antiviral Agents; Brain Chemistry; Cattle; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Microtubules; Protein Conformation; Tubulin

The growth and shortening of microtubules in guanosine triphosphate-(GTP-) mediated dynamic instability has previously been observed to occur at rates which are remarkably variable (Gildersleeve et al., 1992, Chrétien et al., 1995). Neighboring microtubules observed simultaneously can grow or shorten at different rates, and a particular microtubule can undergo changes in rate with the passage of time. This paper addresses the question whether this variability has its origin in processes that involve GTP hydrolysis or whether it results from variations in the structure of microtubules that are independent of GTP hydrolysis. Tubulin was prepared with the nonhydrolyzable GTP analogue GMPPCP [guanylyl 5'-(beta, gamma-methylenediphosphonate)] bound to its exchangeable nucleotide-binding site and with GTP at its nonexchangeable site. Extensive measurements of length changes were obtained by DIC microscopy. Microtubules formed from the GMPPCP tubulin exhibited only growth. No shortening events were observed. Growth occurred at highly variable rates, indistinguishable from those exhibited by GTP tubulin. Subsequent analysis of nucleotides by high-pressure liquid chromatography (HPLC) revealed that some of the GTP that was initially present at the N-site underwent hydrolysis to produce microtubule-bound guanosine diphosphate (GDP). Despite this unexpected finding, one can conclude that variability of growth rate certainly occurs independently of dynamic instability and probably does not involve hydrolysis of GTP at the E-site.

Topics: Animals; Binding Sites; Cattle; Cell-Free System; Guanosine Triphosphate; Hydrolysis; Macromolecular Substances; Microscopy, Interference; Microtubules; Polymers; Tubulin

Microtubules are rigid polymers that contribute to the static mechanical properties of cells. Because microtubules are dynamic structures whose polymerization is regulated during changes in cell shape, we have asked whether the mechanical properties of microtubules might also be modulated. We measured the flexural rigidity, or bending stiffness, of individual microtubules under a number of different conditions that affect the stability of microtubules against depolymerization. The flexural rigidity of microtubules polymerized with the slowly hydrolyzable nucleotide analogue guanylyl-(alpha, beta)-methylene-diphosphonate was 62 +/- 9 x 10(-24) Nm2 (weighted mean +/- SEM); that of microtubules stabilized with tau protein was 34 +/- 3 x 10(-24) Nm2; and that of microtubules stabilized with the antimitotic drug taxol was 32 +/- 2 x 10(-24) Nm2. For comparison, microtubules that were capped to prevent depolymerization, but were not otherwise stabilized, had a flexural rigidity of 26 +/- 2 x 10(-24) Nm2. Decreasing the temperature from 37 degrees C to approximately 25 degrees C, a condition that makes microtubules less stable, decreased the stiffness of taxol-stabilized microtubules by one-third. We thus find that the more stable a microtubule, the higher its flexural rigidity. This raises the possibility that microtubule rigidity may be regulated in vivo. In addition, the high rigidity of an unstabilized, GDP-containing microtubule suggests that a large amount of energy could be stored as mechanical strain energy in the protein lattice for subsequent force generation during microtubule depolymerization.

Topics: Animals; Cattle; Elasticity; Energy Metabolism; Guanosine Triphosphate; Mercaptoethanol; Microscopy; Microtubules; Oxidation-Reduction; Paclitaxel; Pliability; Polymers; Protein Binding; tau Proteins; Tensile Strength; Tubulin

We have used cryoelectron microscopy to try to understand the structural basis for the role of GTP hydrolysis in destabilizing the microtubule lattice. We have measured a structural difference introduced into microtubules by replacing GTP with guanylyl-(alpha,beta)-methylene-diphosphonate (GMPCPP). In a stable GMPCPP microtubule lattice, the moiré patterns change and the tubulin subunits increase in size by 1.5 A. This information provides a clue to the role of hydrolysis in inducing the structural change at the end of a microtubule during the transition from a growing to a shrinking phase.

Topics: Animals; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Microscopy, Fluorescence; Microtubules; Models, Structural; Tubulin

In the present study we found that exocrine pancreatic hyperplasia observed after proximal small bowel resection is accompanied by an increase in pancreatic somatostatin (SS) content at 1 mo and an increase in the number of SS receptors at 2 wk and 1 mo after intestinal surgery. At 6 mo after small bowel resection SS content and SS receptors had returned to control values. However, the original increase in SS receptor number was accompanied by a decrease in the ability of SS to inhibit forskolin-stimulated adenylyl cyclase (AC) activity. In addition, the ability of 5'-guanylylimidodiphosphate (a nonhydrolyzable GTP analogue) to inhibit SS receptor binding was decreased in pancreatic acinar membranes from enterectomized rats at 2 wk and 1 mo after jejunoileal resection. These data suggest that there is an abnormality in the integrity of SS receptor binding site-G protein interactions and would explain the decreased inactivation of AC by SS at 2 wk and 1 mo after proximal small bowel resection.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Colforsin; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hyperplasia; Intestine, Small; Male; Pancreas; Postoperative Period; Rats; Receptors, Somatostatin; Somatostatin; Time Factors

Microtubules polymerized from pure tubulin show the unusual property of dynamic instability, in which both growing and shrinking polymers coexist at steady state. Shortly after its addition to a microtubule end, a tubulin subunit hydrolyzes its bound GTP. Studies with non-hydrolyzable analogs have shown that GTP hydrolysis is not required for microtubule assembly, but is essential for generating a dynamic polymer, in which the subunits at the growing tip have bound GTP and those in the bulk of the polymer have bound GDP. It has been suggested that loss of the 'GTP cap' through dissociation or hydrolysis exposes the unstable GDP core, leading to rapid depolymerization. However, evidence for a stabilizing cap has been very difficult to obtain.. We developed an assay to determine the minimum GTP cap necessary to stabilize a microtubule from shrinking. Assembly of a small number of subunits containing a slowly hydrolyzed GTP analog (GMPCPP) onto the end of dynamic microtubules stabilized the polymer to dilution. By labeling the subunits with rhodamine, we measured the size of the cap and found that as few as 40 subunits were sufficient to stabilize a microtubule.. On the basis of statistical arguments, in which the proportion of stabilized microtubules is compared to the probability that when 40 GMPCPP-tubulin subunits have polymerized onto a microtubule end, all protofilaments have added at least one GMPCPP-tubulin subunit, our measurements of cap size support a model in which a single GTP subunit at the end of each of the 13 protofilaments of a microtubule is sufficient for stabilization. Depolymerization of a microtubule may be initiated by an exposed tubulin-GDP subunit at even a single position. These results have implications for the structure of microtubules and their means of regulation.

Topics: Animals; Biopolymers; Cattle; Guanosine Triphosphate; Microtubules; Tubulin

Measurement of the affinity of microtubules for the anti-cancer drug taxol is problematic, because microtubules are not stable at the very low concentrations required to detect taxol dissociation. We have circumvented this problem by using the GTP analogue GMP-CPP (guanylyl alpha, beta-methylenediphosphonate), which renders microtubules sufficiently stable to allow binding studies with nonsaturating concentrations of taxol. AKd value equal to about 10 nM was estimated from the effect of taxol concentration on the dilution-induced disassembly rate and on the binding of [3H]taxol. With GTP-microtubules the Kd value for taxol binding by tubulin-GDP subunits in the core of the microtubule appears to be comparable with that of GMPCPP-microtubules. However, the stabilizing effect of the drug bound to tubulin subunits that arrive at ends of disassembling microtubules is attenuated by a two-step reaction sequence in which taxol dissociates (k = 30 s-1), followed by rapid (k = 1000 s-1) loss of the taxol-free tubulin subunit. This sequential reaction can be disrupted by high (micromolar) concentrations of taxol, which react rapidly with tubulin subunits at the ends of microtubules (k = 2 x 10(9) M-1 s-1). The inhibitory effect of taxol on microtubule disassembly at concentrations a thousand-fold greater than the Kd value suggests the desirability of using high taxol concentrations in chemotherapy with this compound.

Topics: Animals; Cattle; Guanine Nucleotides; Guanosine Triphosphate; In Vitro Techniques; Microtubules; Paclitaxel; Polymers; Protein Binding; Tubulin

The standard free energy for hydrolysis of the GTP analogue guanylyl-(a,b)-methylene-diphosphonate (GMPCPP), which is -5.18 kcal in solution, was found to be -3.79 kcal in tubulin dimers, and only -0.90 kcal in tubulin subunits in microtubules. The near-zero change in standard free energy for GMPCPP hydrolysis in the microtubule indicates that the majority of the free energy potentially available from this reaction is stored in the microtubule lattice; this energy is available to do work, as in chromosome movement. The equilibrium constants described here were obtained from video microscopy measurements of the kinetics of assembly and disassembly of GMPCPP-microtubules and GMPCP-microtubules. It was possible to study GMPCPP-microtubules since GMPCPP is not hydrolyzed during assembly. Microtubules containing GMPCP were obtained by assembly of high concentrations of tubulin-GMPCP subunits, as well as by treating tubulin-GMPCPP-microtubules in sodium (but not potassium) Pipes buffer with glycerol, which reduced the half-time for GMPCPP hydrolysis from > 10 h to approximately 10 min. The rate for tubulin-GMPCPP and tubulin-GMPCP subunit dissociation from microtubule ends were found to be about 0.65 and 128 s-1, respectively. The much faster rate for tubulin-GMPCP subunit dissociation provides direct evidence that microtubule dynamics can be regulated by nucleotide triphosphate hydrolysis.

Topics: Animals; Brain; Calorimetry; Cattle; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Kinetics; Microscopy, Video; Microtubules; Swine

We have used nuclear magnetic resonance spectroscopy to compare the conformational changes produced by replacement of bound GDP by the GTP analogs guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanylyl (beta, gamma-imido)diphosphate (GMPPNP) in wild-type p21ras as well as the oncogenic mutant (G12D)p21ras. We have used isotope-edited nuclear magnetic resonance spectroscopy to observe the amide resonances of selectively [15N]glycine and [15N]isoleucine labeled p21ras-nucleotide complexes. We find that eight of the nine resonances that respond strongly to GTP gamma S and GMPPNP binding are the same but that the nature of the effect appears different. With GTP gamma S, seven new resonances replace the eight resonances specifically associated with GDP-p21ras, but in GMPPNP-p21ras only two resonances replace the GDP-specific resonances that are lost. The resonance of Gly 60 is clearly shown to be responsive to replacement of GDP by GMPPNP, in addition to glycines 10, 12, 13, 15, and 75 and isoleucines 36, 21, and one other, that were found to respond to GTP gamma S by Miller et al. [Miller, A.-F., Papastavros, M. Z., & Redfield, A.G. (1992) Biochemistry 31, 10208-10216). The two GMPPNP-specific resonances observed appear in positions similar to GTP gamma S-specific resonances, and the GTP gamma S-specific resonances, although not lost altogether, are weaker than the GDP-specific resonances they replace. Thus, the two GTP analogs have similar effects on the spectrum of p21ras, suggesting that the effects are due to features common to both analogs. We propose that active site resonance intensities are specifically attenuated when GTP analogs are bound because interactions with the gamma-phosphate of GTP analogs couple the flexible loops 2 and 4 to the rigid loop 1 of the active site. The conformational heterogeneity and dynamics of loops 2 and 4 would be constrained by loop 1 but also transmitted to it. Coupled conformational exchange on a common intermediate time scale could explain the simultaneous loss of resonances from all three loops in the active site. In our comparison of wild-type and (G12D) GDP-p21ras, we find that the resonance of Ile 36 is not visible in (G12D)p21ras. In (G12D)p21ras, replacement of GDP by GTP gamma S causes the resonances of glycines 10, 13, 15, 60, and 75 and isoleucine 21 and four others to shift from their GDP-specific positions. GTP gamma S-specific resonances are observed for all but two of these.(ABSTRACT TRUNCATED AT

Topics: Binding Sites; Glycine; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Isoleucine; Magnetic Resonance Spectroscopy; Mutation; Protein Conformation; Proto-Oncogene Proteins p21(ras)

The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.

Topics: Animals; Binding Sites; Cattle; Guanosine Triphosphate; Hydrolysis; In Vitro Techniques; Kinetics; Microscopy, Electron; Microtubules; Polymers

In streptolysin O permeabilized acini that were normally responsive to carbamylcholine and cholecystokinin octapeptide, amylase secretion was stimulated: a) by the stable guanyl nucleotides with a potency decreasing as follows: guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) greater than guanylyl-imidodiphosphate (GMP-PNP) = guanylyl-(beta, gamma-methylene)-diphosphonate (GMP-PCP), in the presence of 0.5 mM calcium and b) by calcium alone (EC50 3 microM). The maximal secretory effect of calcium alone (a 2-fold increase) was less effective than that of GTP gamma S and calcium offered in combination (an 8-fold increase). In the virtual absence of Ca2+, GTP gamma S still stimulated amylase release (a 3-fold increase) while 12-O-tetradecanoylphorbol 13-acetate (TPA) did not. The relative potencies of guanyl nucleotides were GTP gamma S greater than GMP-PNP = GMP-PCP = GTP on phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown, GTP gamma S greater than GMP-PNP greater than GMP-PCP = GTP on 45Ca2+ efflux, and GTP GMP-PNP = GMP-PCP = GTP gamma S on [1-14C]arachidonate efflux. Based on these data, the contribution of G proteins to stimulus-secretion coupling beyond the transduction of receptor signal is considered.

Topics: Amylases; Animals; Arachidonic Acid; Bacterial Proteins; Calcium; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylyl Imidodiphosphate; In Vitro Techniques; Inositol Phosphates; Male; Pancreas; Permeability; Rats; Rats, Inbred Strains; Streptolysins; Tetradecanoylphorbol Acetate

Using lead citrate as a capture reagent and adenylate-(beta, gamma-methylene) diphosphate (AMP-PCP) as a substrate, we localized adenylate cyclase activity on the non-ruffled border plasma membrane of approximately half of the osteoclasts on trabecular bone surfaces in the tibial metaphyses of chickens fed a low (0.3%)-calcium diet. The enzyme was not detectable in osteoclasts when chickens were fed a normal calcium diet. Activity was observed on the entire plasma membrane of detached osteoclasts that were situated between osteoblasts on the bone surface and blood vessels in the marrow cavity. Detection of activity on detached osteoclasts required the presence of an activator, implying lower levels in these cells than in those with ruffled borders. Staining was greater on the lateral sides of osteoblasts and osteoclasts when they were in contact with each other. Reaction specificity was indicated by the demonstration of stimulation by forskolin, guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP), dimethylsulfoxide, and NaF, inhibition by alloxan and 2',5'-dideoxyadenosine, and absence of activity when sections were incubated in substrate-free medium or when GMP-PCP replaced AMP-PCP as a substrate. The finding of adenylate cyclase in osteoclast plasma membrane provides structural evidence that the adenylate cyclase-cyclic AMP system has a role in regulation of osteoclast cell function. The low-calcium diet appears to have resulted in increased amounts of adenylate cyclase in osteoclasts.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Animals; Calcium, Dietary; Cell Membrane; Chickens; Guanosine Triphosphate; Histocytochemistry; Osteoclasts

Binding of GTP and GDP to tubulin in the presence or absence of Mg2+ was measured following depletion of the exchangeable site--(E-site) nucleotide. The E-site nucleotide was displaced with a large molar excess of the nonhydrolyzable GTP analogue, GMPPCP, followed by the removal of the analogue. Using a micropartition assay, the equilibrium constant measured in 0.1 M 1.4-piperazinediethanesulfonic acid (Pipes), pH 6.9, 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, 1 mM dithiothreitol, and 1 mM MgSO4 at 4 degrees C was 9.1 x 10(6) M-1 for GTP and 4.4 x 10(6) M-1 for GDP. Removal of Mg2+ reduced the binding affinity of GTP by 160-fold while the affinity of GDP remained essentially unchanged. Similar values were obtained if 0.1 M Tris, pH 7.0, was used instead of Pipes. Binding of Mg2+ to tubulin containing GTP, GDP, or no nucleotide at the E-site was also examined by the micropartition method. Tubulin-GTP contained one high affinity Mg2+ site (K alpha = 1.2 x 10(6) M-1) in addition to the one occupied by Mg2+ as tubulin is isolated, while only weak Mg2+ binding to tubulin-GDP and to tubulin with a vacant E-site (K alpha = 10(3) M-1) was observed. It is suggested that Mg2+ binds to the beta and gamma phosphates of GTP, and only to the beta phosphate of GDP, as shown for the H. ras p21 protein.

Topics: Animals; Binding Sites; Cattle; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Magnesium; Protein Conformation; Tubulin

Free- and EF-2-bound 80 S ribosomes, within the high-affinity complex with the non-hydrolysable GTP analog: guanylylmethylenediphosphonate (GuoPP(CH2)P), and the low-affinity complex with GDP, were treated with trypsin under conditions that modified neither their protein synthesis ability nor their sedimentation constant nor the bound EF-2 itself. Proteins extracted from trypsin-digested ribosomes were unambiguously identified using three different two-dimensional gel electrophoresis systems and 5 S RNA release was checked by submitting directly free- and EF-2-bound 80 S ribosomes, incubated with trypsin, to two-dimensional gel electrophoresis. Our results indicate that the binding of (EF-2)-GuoPP[CH2]P to 80 S ribosomes modified the behavior of a cluster of five proteins which were trypsin-resistant within free 80 S ribosomes and trypsin-sensitive within the high-affinity complex (proteins: L3, L10, L13a, L26, L27a). As for the binding of (EF-2)-GDP to 80 S ribosomes, it induced an intermediate conformational change of ribosomes, unshielding only protein L13a and L27a. Quantitative release of free intact 5 S RNA which occurred in the first case but not in the second one, should be related to the trypsinolysis of protein(s) L3 and/or L10 and/or L26. Results were discussed in relation to structural and functional data available on the ribosomal proteins we found to be modified by EF-2 binding.

Topics: Animals; Electrophoresis, Gel, Two-Dimensional; Guanosine Diphosphate; Guanosine Triphosphate; Liver; Models, Biological; Peptide Elongation Factor 2; Peptide Elongation Factors; Phosphoproteins; Protein Binding; Rats; Ribosomal Proteins; Ribosomes; Trypsin

The accessibility of three amino acids of EF-2, located within highly conserved regions near the N- and C-terminal extremities of the molecule (the E region and the ADPR region, respectively) to modifying enzymes has been compared within nucleotide-complexed EF-2 and ribosomal complexes that mimic the pre- and posttranslocational ones: the high-affinity complex (EF-2)-nonhydrolysable GTP analog GuoPP[CH2]P ribosome and the low-affinity (EF-2)-GDP-ribosome complex, EF-2 and ribosomes being from rat liver. We studied the reactivity of two highly conserved residues diphthamide-715 and Arg-66, to diphtheria-toxin-dependent ADP-ribosylation and trypsin attack, and of a threonine that probably lies between residues 51 and 60, to phosphorylation by a Ca2+/calmodulin-dependent protein kinase. Diphthamide 715 and this threonine residue were unreactive within the high-affinity complex but seemed fully reactive in the low-affinity complex. Arg-66 was resistant to trypsin in both complexes. The possible involvement of the E and ADPR regions of EF-2 in the interaction with ribosome in the two complexes is discussed.

Topics: Adenosine Diphosphate Ribose; Animals; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Liver; Mutation; NAD; Peptide Elongation Factor 2; Peptide Elongation Factors; Peptide Mapping; Phosphoproteins; Phosphorylation; Protein Binding; Rats; Ribosomes; Trypsin

Complete replacement of the nucleotide on the exchangeable binding site of purified calf brain tubulin by the non-hydrolyzable GTP-analogue guanylyl-(beta,gamma-methylene)diphosphonate (GMPPCP) has been achieved by treatment of tubulin-GDP with phosphodiesterase-free alkaline phosphatase. GMPPCP binds to tubulin with a low affinity relative to GTP or GDP. Binding of the analogue is linked to magnesium ion concentration and, like the binding of other guanine nucleotides, is promoted by high concentrations of glycerol. The complex of pure tubulin and GMPPCP readily assembles at 37 degrees C into microtubules or curled ribbons of protofilaments, depending on buffer composition. Assemblies are cold-reversible at 0-2 degrees C, and multiple reversible assemblies can be observed during repeated heating/cooling cycles.

Topics: Animals; Brain; Cattle; Chromatography, High Pressure Liquid; Glycerol; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Microscopy, Electron; Protein Binding; Tubulin

Studies on a variety of animal cell types have revealed a GTP-specific calcium-releasing mechanism in a non-mitochondrial, microsomal fraction. Here we report that GTP also induces rapid release of calcium from a zucchini (Cucurbita pepo L.) hypocotyl microsomal fraction. Maximal release occurs at 50 microM, and half-maximal release at 8 microM GTP. GTP is highly specific in its effect, and may not be replaced by UTP, ATP, CTP, TTP, GMP, or by non-hydrolysable analogues of GTP. Reuptake of calcium after release does not normally occur; however, this may be induced by non-hydrolysable GTP analogues. Calcium release is also blocked by prior treatment with these analogues.

Topics: Adenosine Triphosphate; Calcium; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Microscopy, Electron; Microsomes; Plants; Polyethylene Glycols; Thionucleotides

The role of a guanine nucleotide-binding protein (Gk) in the coupling between muscarinic receptor activation and opening of an inwardly rectifying K+ channel [IK(M)] was examined in cardiac atrial myocytes, using hydrolysis-resistant GTP analogues. In the absence of muscarinic agonist, GTP analogues produced a membrane current characteristic of IK(M). The initial rate of appearance of this receptor-independent IK(M) was measured for the various analogues in order to explore the kinetic properties of IK(M) activation. We found that IK(M) activation is controlled solely by the intracellular analogue/GTP ratio and not by the absolute concentrations of the nucleotides. Analogues competed with GTP for binding to Gk with the following relative affinities: GTP gamma S greater than GTP greater than GppNHp greater than GppCH2p. At sufficiently high intracellular concentrations, however, all GTP analogues produced the same rate of IK(M) activation. This analogue-independent limiting rate is likely to correspond to the rate of GDP release from inactive, GDP-bound Gk. Muscarinic receptor stimulation by nanomolar concentrations of acetylcholine (ACh), which do not elicit IK(M) under control conditions, catalyzed IK(M) activation in the presence of GTP analogues. The rate of Gk activation by ACh (kACh) was found to be described by the simple relationship kACh = 8.4 X 10(8) min-1 M-1.[ACh] + 0.44 min-1, the first term of which presumably reflects the agonist-catalyzed rate of GDP release from the Gk.GDP complex, while the second term corresponds to the basal rate of receptor-independent GDP release. Combined with the estimated K0.5 of the IK(M)-[ACh] dose-effect relationship, 160 nM, this result also allowed us to estimate the rate of Gk.GTP hydrolysis, kcat, to be near 135 min-1. These results provide, for the first time, a quantitative description of the salient features of G-protein function in vivo.

Topics: Acetylcholine; Animals; Cells, Cultured; Electrophysiology; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylyl Imidodiphosphate; Heart Atria; Ion Channels; Kinetics; Myocardium; Potassium; Rana catesbeiana; Receptors, Muscarinic; Thionucleotides

Isolated human polymorphonuclear (PMN) leukocyte plasma membranes express high affinity (mean Kd = 0.12 nM) and low affinity (mean Kd = 50 nM) receptors for the chemotactic factor leukotriene B4 (5(S),12(R)-dihydroxy-eicosa-6,14 cis-8,10 trans-tetraenoic acid; LTB4) that are similar to those on intact PMN leukocytes. A portion of high affinity LTB4-R on PMN leukocyte membranes were converted to the low affinity state by GTP (mean +/- SE = 28.6 +/- 14.0%) and nonhydrolyzable GTP analogues, such as 5'-guanylylimidodiphosphate (GMP-PNP), in a concentration-dependent, nucleotide-specific, and reversible manner, without altering the intrinsic binding affinities of either class. [3H]GMP-PNP bound specifically to one class of receptors (mean Kd = 13 nM) on PMN leukocyte membranes. The interdependence of the LTB4-binding membrane protein and guanine nucleotide-binding protein was suggested by the capacity of LTB4 to enhance by a maximum of 150% the binding of [3H]GMP-PNP to PMN leukocyte membranes by increasing the number, but not altering the affinity, of receptors for GMP-PNP. Pertussis toxin, but not cholera toxin, reversed the enhancement of binding of [3H]GMP-PNP produced by LTB4. Guanine nucleotide-binding proteins and high affinity LTB4-R thus exhibit a mutual regulation that differs mechanistically from that of peptide chemotactic factor receptors on PMN leukocytes.

Topics: Cell Membrane; Cholera Toxin; Cyclic GMP; Guanosine Monophosphate; Guanosine Triphosphate; Humans; Leukotriene B4; Neutrophils; Pertussis Toxin; Receptors, Immunologic; Receptors, Leukotriene B4; Virulence Factors, Bordetella

Monospecific polyclonal antibodies against seven proteins of the 40 S subunit of rat liver ribosomes were used to identify ribosomal proteins involved in interaction with initiation factor eIF-2 in the quaternary initiation complex [eIF-2 X GMPPCP X [3H]Met-tRNAf X 40 S ribosomal subunit]. Dimeric immune complexes of 40 S subunits mediated by antibodies against ribosomal proteins S3a, S13/16, S19 and S24 were found to be unable to bind the ternary initiation complex [eIF-2 X GMPPCP X [3H]Met-tRNAf]. In contrast, 40 S dimers mediated by antibodies against proteins S2, S3 and S17 were found to bind the ternary complex. Therefore, from the ribosomal proteins tested, only proteins S3a, S13/16, S19 and S24 are concluded to be involved in eIF-2 binding to the 40 S subunit.

Topics: Animals; Centrifugation, Density Gradient; Cross-Linking Reagents; Eukaryotic Initiation Factor-2; Guanosine Triphosphate; Immunoassay; Liver; Macromolecular Substances; Peptide Initiation Factors; Proteins; Rats; Ribosomal Proteins; Ribosomes; RNA, Transfer, Met

Removal of GDP from tubulin E-site is not obligatory for the in vitro assembly of microtubule protein in 0.5 mM GMPPCP. This assembly, which is significantly enhanced by glycerol, produces microtubules of normal morphology and with normal composition of microtubule-associated proteins (MAPs). [3H]-GDP initially present at the E-site is shown to be incorporated directly into microtubules during assembly; this incorporation, maximally 60% of the assembled polymer, is dependent upon MAPs. These results are consistent with oligomeric species composed principally of GDP-tubulin plus MAPs, being incorporated directly into microtubules. The finding that stoichiometric GTP-tubulin formation is not an essential prerequisite for microtubule assembly may have important implications for the energetics of microtubule formation.

Topics: Animals; Cattle; Diphosphonates; Glycerol; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Monophosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Microscopy, Electron; Microtubule-Associated Proteins; Microtubules; Tubulin

Guanine nucleotides are successfully used in the studies of regulatory N-proteins coupled with adenylate cyclase. In the present work N-chloroacetylhydrazones of oxo-GTP and oxo-GDP are described. After 4 hr preincubation of these nucleotides with plasma membranes from bovine brain caudate nucleus, the ability of adenylate cyclase to be activated by guanylyl-5'-methylene-diphosphonate is blocked. The degree of inhibition depends on preincubation time and increases in the presence of Mg2+. Guanylyl-5'-methylenediphosphonate protects adenylate cyclase from the action of N-chloroacetylhydrazone of oxo-GTP. These findings suggest that adenylate cyclase activation is diminished as a result of covalent modification of the Ns. N-chloroacetyl-hydrazone of oxo-GDP also causes a loss of the adenylate cyclase sensitivity to the fluoride ion and cholera toxin.

Topics: Adenylyl Cyclase Inhibitors; Animals; Cattle; Caudate Nucleus; Diphosphonates; Enzyme Activation; GTP-Binding Proteins; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Monophosphate; Guanosine Triphosphate; In Vitro Techniques; Magnesium

The different functional complexes of ribosomes with elongation factor F (EF-G) were studied by digestion experiments with trypsin. It was found that upon interaction of EF-G with ribosomes the L7/L12 proteins are sensitive to trypsin and are trypsin resistant after dissociation of EF-G from ribosomes. The significance of conformational alterations in the L7/L12 and also in the other proteins in the translation process is discussed.

Topics: Bacterial Proteins; Diphosphonates; Escherichia coli; Fusidic Acid; Guanosine Diphosphate; Guanosine Monophosphate; Guanosine Triphosphate; Peptide Elongation Factor G; Peptide Elongation Factors; Protein Conformation; Ribosomal Proteins; Ribosomes; Trypsin

The effect of certain antiviral compounds said to act because of an increased permeability of virally infected cells has been tested in SFV-infected BHK cells. The time at which SFV-infected BHK cells become sensitive to the action of GppCH2p is more than an hour later than the time at which protein synthesis in such cells becomes depressed. The uptake of [3H]GppCH2p is the same in infected and uninfected cells, whether measured at 19 or 37 degrees C. We conclude that GppCH2p, and probably other 'impermeant' inhibitors of protein synthesis also, affect virally-infected cells selectively not because of an increased permeability, but because of a general impairment of protein synthesis in such cells.

Topics: Animals; Antiviral Agents; Cell Line; Cell Membrane Permeability; Cricetinae; Diphosphonates; Guanine Nucleotides; Guanosine Monophosphate; Guanosine Triphosphate; Kidney; Protein Biosynthesis; Semliki forest virus; Togaviridae Infections; Virus Diseases

tRNAPhe and tRNAVal of Escherichia coli were derivatized at the S4U8 position with p-azidophenacyl and p-azidophenacylacetate photoaffinity probes. The modified tRNAs could still function efficiently in all of the partial reactions of protein synthesis except for an approximately sevenfold decrease in the rate of translocation. Irradiation (310 to 340 nm) of probe-modified Phe-tRNA or Val-tRNA placed in the ribosomal A site led to crosslinking that was totally dependent on irradiation, the presence of the azido group on the probe, mRNA, and elongation factor Tu (EFTu). Prephotolysis of the modified tRNA abolished crosslinking, but prephotolysis of the ribosomes and factors had little effect. Crosslinking was efficiently quenched by mercaptoethanol or dithiothreitol, demonstrating accessibility of the probe to solvent. Use of GDPCP in place of GTP also blocked crosslinking, probably because of the retention of EFTu on the ribosome. Crosslinking with the p-azidophenacyl acetate (12 A) probe was only half as efficient as with the p-azidophenacyl (9 A) probe, and this ratio was not changed by varying Mg2+ from 5 to 15 mM. The crosslink was from a functional A site, since AcPhePhe-tRNA at the A site could be crosslinked, and it was A site-specific, because neither translocation nor direct non-enzymatic P site binding yielded any significant covalent product. The crosslink was to ribosomal protein(s) of the 30 S subunit. No other ribosomal component was crosslinked. Identification of the protein crosslinked is described in the accompanying paper.

Topics: Affinity Labels; Azides; Binding Sites; Diphosphonates; Escherichia coli; Guanosine Monophosphate; Guanosine Triphosphate; Kinetics; Magnesium; Mercaptoethanol; Peptide Elongation Factor Tu; Peptide Elongation Factors; Photolysis; Ribosomes; RNA, Transfer, Amino Acyl; Thiouridine

Addition of 10 microM guanyl-5'-ylimidodiphosphate at 30 degrees or 0 degree to guinea pig brain particulates instantaneously evoked nearly 50% inhibition of adenylate cyclase activity as determined after removal of the GTP analog by washing of the particulates. The inhibitory state, once formed, persisted for at least 60 min as long as the preparation was kept either in a medium devoid of the analog (0-30 degrees) or in its presence at 0 degree. During incubation at 30 degrees in the presence of the analog, however, the inhibited or nontreated enzyme showed a gradual increase in enzyme activity. Both the inhibitory and the activating effects of the analog were saturable, with a half-maximal concentration of about 1.0 microM, and were antagonized by simultaneous addition of GTP, GDP, and GMP (in decreasing order). The persistently inhibited enzyme enabled the detection of marked stimulation by norepinephrine and histamine, whereas these amines showed only marginal stimulation of the enzyme before treatment with the analog. Formation of such a persistent inhibitory state appears to be specific to brain cyclase.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Imidodiphosphate; Animals; Brain; Diphosphonates; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Monophosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Guinea Pigs; Histamine; Magnesium; Male; Manganese; Norepinephrine; Thionucleotides

The uptake of the GTP analogue guanylyl(beta,gamma-methylene)diphosphonate (GppCH2p) is the same in Semliki Forest virus (SFV)-infected BHK cells as in mock-infected cells, in spite of the fact that protein synthesis is inhibited by GppCH2p more markedly in SFV-infected cells than in control cells. A possible explanation for this difference is that infected cells have a lower concentration of GTP and a lower ratio of GTP:GDP than uninfected cells, and the analogue may thus be a more effective competitive inhibitor of translation. We conclude that in this system, the difference between infected and uninfected cells lies not at the plasma membrane but within the cytoplasm.

Topics: Animals; Cell Line; Cell Membrane Permeability; Cricetinae; Diphosphonates; Guanine Nucleotides; Guanosine Monophosphate; Guanosine Triphosphate; Methylglucosides; Protein Biosynthesis; Semliki forest virus

Topics: Adenylyl Cyclases; Animals; Diphosphonates; Enzyme Activation; Female; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Monophosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Muscle, Smooth; Rats; Thionucleotides

D-Ala2-Met5-enkephalin, morphine, and noradrenaline inhibit the adenylate cyclase in homogenates of neuroblastoma x glioma hybrid cells in a dose-dependent manner even after the enzyme has been preactivated by cholera toxin. Half-maximal inhibition and extent of inhibition are the same with native or cholera toxin-activated enzyme. The inhibition caused by opioids or noradrenaline are antagonized by naloxone or phentolamine, respectively. The effect of D-Ala2-Met5-enkephalin on cholera toxin-activated enzyme is immediate in onset and rapidly reversed by the addition of naloxone. Guanyl-5'-yl-imidodiphosphate stimulates basal activity but inhibits the enzyme activated by cholera toxin or prostaglandin E1. Stimulation occurs at a concentration of 100 microM or above, inhibition even at 0.1 microM. The inhibitory effect of the non-hydrolysable GTP analog is antagonized by GTP. Guanyl-5'-yl-methylenediphosphonate, another nonhydrolysable GTP analog, inhibits basal as well as cholera toxin-stimulated or prostaglandin E1-stimulated adenylate cyclase. Other guanine derivatives such as GDP, GMP, cyclic GMP, guanyl-5'-yl-phosphoric acid amide and guanosine have no effect under the same conditions. The results may be taken as a piece of evidence for two separate guanyl nucleotide-binding sites accompanying the adenylate cyclase in the hybrid cells and mediating, respectively, stimulation and inhibition of the enzyme by hormones.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cholera Toxin; Clone Cells; Diphosphonates; Endorphins; Enkephalin, Methionine; Enkephalins; Glioma; Guanosine Monophosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Mice; Morphine; Neuroblastoma; Norepinephrine; Rats

Topics: Animals; Cell Membrane Permeability; Complement System Proteins; Cytarabine; Diphosphonates; Erythrocyte Membrane; Guanosine Monophosphate; Guanosine Triphosphate; Lymphoma; Mice; Osmolar Concentration; Rabbits; Sucrose

The infection of animal cells by encephalomyocarditis (EMC) virus lead to a drastic change in membrane permeability towards low mol. wt. compounds. Addition of the nucleotide analogue GppCH2p to the culture medium resulted in a specific inhibition of protein synthesis in EMC virus-infected 3T6 cells. This inhibition was not observed when GTP or ATP were present nor in control mock-infected 3T6 cells. The induction of membrane leakiness after viral infection was not specific for 3T6 cells, as it was also detected in mouse L cells, hamster BHK-21 cells and monkey CV1 cells. The inhibitory action produced by GppCH2p in virus-infected cells was fully reversed upon addition of fresh medium. Moreover, analysis of the proteins synthesized after medium replacement showed a preferential synthesis of cellular proteins. The presence of zinc ions resulted in an inhibition of the cleavage of large viral polypeptide precursors to mature viral proteins. Under these conditions, membrane leakiness as measured by GppCH2p, was not observed. However, this seems to be an effect of zinc ions themselves on the membrane, because inhibition of mature protein formation by other means, such as the presence of amino acid analogues, did not prevent inhibition of translation by GppCH2p in virus-infected cells. Addition of the cap analogues 7mGppp and 2'-O'-mGppp, resulted in specific stimulation of viral protein synthesis in EMC virus-infected 3T6 cells. On the other hand, the presence of 7mGp had no effect on translation. We propose that a specific capping of viral mRNA takes place in the presence of these compounds, and leads to increased stability and greater efficiency in the translation of viral mRNA.

Topics: Diphosphonates; Encephalomyocarditis virus; Guanosine Monophosphate; Guanosine Triphosphate; Protein Biosynthesis; RNA Cap Analogs; RNA Caps

Topics: Animals; Brain; Brain Chemistry; Diphosphonates; Guanine Nucleotides; Guanosine Monophosphate; Guanosine Triphosphate; Kinetics; Macromolecular Substances; Microscopy, Electron; Microtubules; Phosphates; Swine; Tubulin

We have examined the properties of microtubules formed in the presence of GTP, 5'-guanylyl imidodiphosphate (GMPP(NH)P), and 5'-guanylyl methylenediphosphate (GMPP(CH2)P) to identify features of the assembly or disassembly reactions uniquely related to hydrolysis. The assembly of microtubules with GTP or GMPP(NH)P was similar in terms of rates and extents of assembly, the length distributions, and podophyllotoxin-induced depolymerization. The greater rapidity of GMPP(CH2)P-supported assembly, however, resulted in shorter, more numerous microtubules and the rate of podophyllotoxin-induced depolymerization was consistent with an increased number of concentration of microtubules. Experiments with GTP or analogue incorporation and release indicated that GTP-tubule turnover corresponded to a rate of about 8% of the microtubule protein taken up or released per h. With GMPP(NH)P- and GMPP(CH2)P-tubules, the rates of label uptake by unlabeled microtubules were considerably lower than observed with guanosine triphosphate. We suggest that exchange experiments can reflect contributions from head-to-tail polymerization and polymer length redistribution, but it is not as yet possible to evaluate the relative contributions of each process.

Topics: Animals; Brain; Cattle; Diphosphonates; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Monophosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Macromolecular Substances; Microtubules; Podophyllotoxin; Tubulin