guanosine-diphosphate and beryllium-fluoride

The fusion of mammalian inner mitochondrial membranes (IMMs) is mediated by dynamin-like GTPase OPA1. Mutations in human OPA1 cause optic atrophy, but the molecular basis for membrane fusion and pathogenesis is not clear. Here, we determined the crystal structure of the minimal GTPase domain (MGD) of human OPA1. A three-helix bundle (HB) domain including two helices extending from the GTPase (G) domain and the last helix of OPA1 tightly associates with the G domain. In the presence of GDP and BeF3-, OPA1-MGD forms a dimer, the interface of which is critical for the maintenance of mitochondrial morphology. The catalytic core of OPA1 possesses unique features that are not present in other dynamin-like proteins. Biochemical experiments revealed that OPA1-MGD forms nucleotide-dependent dimers, which is important for membrane-stimulated GTP hydrolysis, and an N-terminal extension mediates nucleotide-independent dimerization that facilitates efficient membrane association. Our results suggest a multifaceted assembly of OPA1 and explain the effect of most OPA1 mutations on optic atrophy.

Topics: Beryllium; Binding Sites; Cloning, Molecular; Crystallography, X-Ray; Escherichia coli; Fluorides; Gene Expression; Genetic Vectors; GTP Phosphohydrolases; Guanosine Diphosphate; Humans; Liposomes; Magnesium; Models, Molecular; Mutation; Optic Atrophy; Potassium; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Multimerization; Recombinant Proteins

The size and chemical nature of the stabilizing cap at microtubule (MT) ends has remained enigmatic, in large part because it has been difficult to detect and measure it directly. By pulsing steady-state suspensions of bovine brain microtubules (MTs) with trace quantities of [gamma(32)P]GTP and sedimenting the MTs through 50% sucrose cushions to reduce background contaminating (32)P to negligible levels, we were able to detect a small number of (32)P molecules that remain stably bound to the MTs (a mean of 25.5 molecules of (32)P per MT). Analysis of the chemical form of the stably bound (32)P by thin-layer chromatography revealed that it was all (32)P-orthophosphate ((32)P(i)). The (32)P(i) was determined to be located at the MT ends because colchicine and vinblastine, drugs that suppress tubulin incorporation into the MT by binding specifically at MT ends, reduced the quantity of the stably bound (32)P(i). Taxol, a drug that stabilizes MT dynamics by binding along the MT surface rather than at the ends, did not affect the stoichiometry of the bound (32)P(i). If the bound (32)P is equally distributed between the two ends, each end would contain 12-13 molecules of (32)P(i). Beryllium fluoride (BeF(3-)) and aluminum fluoride (AlF(4-)), inorganic phosphate analogues, suppressed the dynamic instability behavior of individual MTs and, thus, stabilized them. For example, BeF(3-) (70 microM) reduced the MT shortening rate by 2.5-fold and decreased the transition frequency from the growing or the attenuated state to rapid shortening by 2-fold. The data support the hypothesis that the stabilizing cap at MT ends consists of a single layer of tubulin GDP-P(i) subunits. The data also support the hypothesis that the mechanism giving rise to the destabilized GDP-tubulin core involves release of P(i) rather than hydrolysis of the GTP.

Topics: Aluminum Compounds; Animals; Beryllium; Binding Sites; Brain Chemistry; Cattle; Fluorides; Guanosine Diphosphate; Guanosine Triphosphate; Male; Microscopy, Video; Microtubules; Phosphates; Phosphorus Radioisotopes; Polymers; Sea Urchins; Spermatozoa; Thermodynamics; Tubulin

The role of the switch II region in the conformational transition of activation of Ha-ras-p21 has been investigated by mutating residues predicted to act as hinges for the conformational transition of this loop (Ala59, Gly60, and Gly75) (Díaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997, Proteins 28:434-451), as well as mutating the catalytic residue Gln61. The proposed mutations of the hinge residues decrease the rate of the conformational transition of activation as measured by the binding of BeF3- to the GDP-p21 complex. Also, the thermodynamic parameters of the binding reaction are altered by a factor between three and five, depending on the temperature. (Due to changes in activation and reaction enthalpies, partially compensated by entropy changes.) The control mutation Q61H in which only the catalytic residue is changed has only a limited effect on the kinetic rate constants of the conformational transition and on the thermodynamic parameters of the reaction. The fact that mutations of the hinge residues of the switch II region affect both the binding of the phosphate analog and the conformational transition of activation indicates that the switch II is implicated both in the early and the late states of the transition.

Topics: Beryllium; Binding Sites; Catalytic Domain; Fluorides; Guanosine Diphosphate; Humans; In Vitro Techniques; Kinetics; Mutagenesis, Site-Directed; Protein Conformation; Proto-Oncogene Proteins p21(ras); Recombinant Proteins; Thermodynamics

The proposal that microtubule dynamic instability results from stabilization of microtubule ends by tubulin-GDP-Pi subunits (where Pi is inorganic phosphate) [Melki et al. (1996) Biochemistry 35, 12038] was based on studies of GTP hydrolysis and microtubule assembly that showed that tubulin-GDP-Pi subunits can transiently accumulate at microtubule ends. There is no direct evidence that GDP-Pi-subunits can stabilize microtubules under conditions where dynamic instability is observed and this has been inferred from the observation that tubulin-GDP-BeFn subunits stabilize microtubules. To test if tubulin-GDP-Pi stabilizes microtubules we sought evidence for a synergism between the effect of Pi and BeFn. We found, however, that Pi antagonizes the effect of BeFn by displacing it from tubulin subunits. The alternate mechanism in which Pi inhibits BeFn stabilization of microtubules by displacing fluoride from beryllium was ruled out from the 9Be and 19F NMR spectra in the presence and absence of Pi. Further evidence that tubulin-GDP-BeFn is not an analogue of tubulin-GDP-Pi and that tubulin-GDP-Pi is not responsible for maintaining the growth phase in microtubules manifesting dynamic instability was provided by our observation that Pi did not decrease the disassembly rate under conditions where tubulin-GDP-Pi subunits are expected to have formed. Results showing that BeFn binds randomly to subunits in microtubules provided evidence that Pi dissociation from the tubulin-GDP-Pi intermediate formed during GTP hydrolysis occurs randomly rather than processively starting at the growing microtubule tip.

Topics: Animals; Beryllium; Cattle; Fluorides; Guanosine Diphosphate; Microtubules; Models, Biological; Phosphates; Protein Binding; Protein Processing, Post-Translational; Tubulin

Hitherto ras-related GTP-binding proteins have been considered not to bind phosphate analogs (Kahn, R. A. (1991) J. Biol. Chem. 266, 15595-15597), at least in the absence of activating proteins (Mittal, R., Reza, M., Goody, R., and Wittinghofer, A. (1996) Science 273, 115-117). In this work, we have used a fluorescent active mutant (Y32W) of p21(Ha-)ras to demonstrate that BeF3- binds to the GDP. p21(Ha-ras) complex in the absence of activating proteins. It induces a conformational change leading to a state with fluorescence properties similar to those of the active state. The binding has a low affinity (Kd at 25 degrees C = 8.1 +/- 0.3 mM) and is endothermic (DeltaH = 22.3 +/- 1.6 kJ mol-1). The similarity between the GTP-bound form and the GDP.BeF3--bound form has been confirmed using lifetime analysis of the tryptophan fluorescence. The kinetic analysis of the process indicates that the binding can be divided into a first bimolecular step, which accounts for the association of the anion with its binding site, and a second step, which corresponds to an internal conformational transition of the GDP. BeF3-.p21(Ha-)ras complex to its final state. Both steps are endothermic (DeltaH1 = 15 +/- 2 kJ mol-1 and DeltaH2 = 8 +/- 2 kJ mol-1). The kinetically determined enthalpy change of 23 +/- 4 kJ mol-1 is in excellent agreement with the equilibrium analysis.

Topics: Anions; Beryllium; Flow Injection Analysis; Fluorides; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Models, Chemical; Mutation; Phosphates; Protein Conformation; Proto-Oncogene Proteins p21(ras); Recombinant Proteins; Spectrometry, Fluorescence

It has been recently shown by differential scanning calorimetry (DSC) that the formation of stable complexes of myosin subfragment 1 (S1) with Mg-ADP and orthovanadate (Vi) or beryllium fluoride (BeFx) causes a global conformational change in the S1 molecule which is reflected in a pronounced increase of S1 thermal stability and in a significant change of S1 domain structure [Shriver, J. W., & Kamath U. (1990) Biochemistry 29, 2556-2564; Levitsky, D. I., Shnyrov, V. L., Khvorov, N. V., Bukatina, A. E., Vedenkina, N. S., Permyakov, E. A., Nikolaeva, O. P., & Poglazov, B. F. (1992) Eur. J. Biochem. 209, 829-835; Bobkov, A. A., Khvorov, N. V., Golitsina, N. L., & Levitsky, D. I. (1993) FEBS Lett. 332, 64-66]. In this work, which continues the previous investigations, we report on a DSC study of the complexes of S1 with various nucleoside diphosphates (NDP). In the absence of Vi or BeFx the various Mg(2+)-NDP and Mg(2+)-PPi had a similar effect on the S1 conformation. All of them had practically no influence on the temperature of the thermal transition but increased its sharpness. However, in the presence of Vi or BeFx the effects of Mg(2+)-NDP complexes were quite different from each other and strongly depended on the base structure of NDP; their effectiveness in inducing conformational changes in S1 and the stability of these complexes decreased in the following order: ADP > CDP >> UDP >> IDP > GDP.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Diphosphate; Beryllium; Calorimetry, Differential Scanning; Cytidine Diphosphate; Fluorides; Guanosine Diphosphate; Inosine Diphosphate; Myosin Subfragments; Nucleotides; Protein Conformation; Thermodynamics; Uridine Diphosphate; Vanadates

Beryllium fluoride (BeF3-) has previously been shown to bind tightly to microtubules as a structural analogue of Pi and to mimic the GDP-Pi transient state in tubulin polymerization [Carlier, M.-F., Didry, D., Melki, R., Chabre, M., & Pantaloni, D. (1988) Biochemistry 27, 3555-3559]. The interaction of BeF3- with tubulin is analyzed here in greater detail. BeF3- binds to and dissociates from microtubule GDP subunits at very slow rates (k+ congruent to 100 M-1 s-1; k- congruent to 6 x 10(-4) s-1), suggesting that a slow conformation change of tubulin, linked to the stabilization of the microtubule structure, follows BeF3- binding. The possibility is evoked that BeF3- acts as a transition-state analogue in the GTPase reaction of tubulin. BeF3- does not bind to dimeric nor to oligomeric GDP-tubulin with high affinity. Substoichiometric binding of BeF3- to microtubules provides extensive stabilization of the structure. An original mechanistic model that accounts for the data is proposed. The kinetic parameters for microtubule elongation in the presence of GTP- and GDP-tubulin with and without BeF3- have been determined. Data support the following views: (i) Microtubules at steady state and in a regime of slow growth in the presence of GTP are stabilized by a cap of GDP-Pi subunits functionally similar to GDP-BeF3 subunits. (ii) In the presence of BeF3-, microtubules elongate from GDP-tubulin within the following sequence of reactions: initial nonproductive binding of GDP-tubulin to microtubule ends is followed by the binding of BeF3- and the associated conformation change allowing sustained elongation.

Topics: Animals; Beryllium; Brain; Fluorides; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Kinetics; Macromolecular Substances; Mathematics; Microtubules; Models, Theoretical; Phosphates; Structure-Activity Relationship; Swine; Tubulin

Aluminum fluoride and beryllium fluoride complexes have previously been shown to bind tightly to F-ADP-actin and GDP-microtubules in competition with Pi and to mimic the XDP-Pi transient state of the polymerization. The structure of the bound complexes is investigated here in further detail. Using a fluoride ion-specific electrode, the number of fluoride atoms per aluminum or beryllium atom in the bound complex could be determined. The results indicate that AIF-4 and either BeF2(OH)-.H2O or BeF3-.H2O are the tightly bound species in both F-actin and microtubules. The dependences of the binding on pF and pH are consistent with this conclusion. The possible geometries of aluminum and beryllium fluorides in the gamma-phosphate subsite of the nucleotide are discussed in correlation with the catalytic mechanism of nucleotide hydrolysis.

Topics: Actins; Adenosine Diphosphate; Aluminum; Aluminum Compounds; Animals; Beryllium; Fluorides; Guanine Nucleotides; Guanosine Diphosphate; Microtubules; Muscles; Rabbits

Fluoride activation of G proteins requires the presence of aluminium or beryllium and it has been suggested that AIF4- acts as an analogue of the gamma-phosphate of GTP in the nucleotide site. We have investigated the action of AIF4- or of BeF3- on transducin (T), the G protein of the retinal rods, either indirectly through the activation of cGMP phosphodiesterase, or more directly through their effects on the conformation of transducin itself. In the presence of AIF4- or BeF3-, purified T alpha subunit of transducin activates purified cyclic GMP phosphodiesterase (PDE) in the absence of photoactivated rhodopsin. Activation is totally reversed by elution of fluoride or partially reversed by addition of excess T beta gamma. Activation requires that GDP or a suitable analogue be bound to T alpha: T alpha-GDP and T alpha-GDP alpha S are activable by fluorides, but not T alpha-GDP beta S, nor T alpha that has released its nucleotide upon binding to photoexcited rhodopsin. Analysis of previous works on other G proteins and with other nucleotide analogues confirm that in all cases fluoride activation requires that a GDP unsubstituted at its beta phosphate be bound in T alpha. By contrast with alumino-fluoride complexes, which can adopt various coordination geometries, all beryllium fluoride complexes are tetracoordinated, with a Be-F bond length of 1.55 A, and strictly isomorphous to a phosphate group. Our study confirms that fluoride activation of transducin results from a reversible binding of the metal-fluoride complex in the nucleotide site of T alpha, next to the beta phosphate of GDP, as an analogue of the gamma phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Aluminum; Aluminum Compounds; Animals; Beryllium; Cattle; Fluorides; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Photoreceptor Cells; Protein Binding; Rod Cell Outer Segment