guanosine-monophosphate and 2--guanylic-acid

The ribonuclease T1 variant 9/5 with a guanine recognition segment, altered from the wild-type amino acid sequence 41-KYNNYE-46 to 41-EFRNWQ-46, has been cocrystallised with the specific inhibitor 2'-GMP. The crystal structure has been refined to a crystallographic R factor of 0.198 at 2.3 A resolution. Despite a size reduction of the binding pocket, pushing the inhibitor outside by 1 A, 2'-GMP is fixed to the primary recognition site due to increased aromatic stacking interactions. The phosphate group of 2'-GMP is located about 4.2 A apart from its position in wild-type ribonuclease T1-2'-GMP complexes, allowing a Ca(2+), coordinating this phosphate group, to enter the binding pocket. The crystallographic data can be aligned with the kinetic characterisation of the variant, showing a reduction of both, guanine affinity and turnover rate. The presence of Ca(2+) was shown to inhibit variant 9/5 and wild-type enzyme to nearly the same extent.

Topics: Amino Acid Sequence; Amino Acid Substitution; Binding Sites; Calcium; Crystallization; Crystallography, X-Ray; Escherichia coli; Genetic Variation; Guanine; Guanosine Monophosphate; Hydrogen Bonding; Hydrolysis; Kinetics; Models, Molecular; Molecular Sequence Data; Phosphates; Protein Conformation; Ribonuclease T1; RNA; Substrate Specificity; Water

The carboxymethylation of RNase T1 at the gamma-carboxyl group of Glu58 leads to a complete loss of the enzymatic activity while it retains substrate-binding ability. Accompanying the carboxymethylation, RNase T1 undergoes a remarkable thermal stabilization of 9 degrees C in the melting temperature (Tm). In order to clarify the inactivation and stabilization mechanisms of RNase T1 by carboxymethylation, the crystal structure of carboxymethylated RNase T1 (CM-RNase T1) complexed with 2'-GMP was determined at 1.8 A resolution. The structure, including 79 water molecules and two Na+, was refined to an R factor of 0.194 with 10 354 reflections > 1 sigma (F). The carboxyl group of CM-Glu58, which locates in the active site, occupies almost the same position as the phosphate group of 2'-GMP in the crystal structure of intact RNase T1.2'-GMP complex. Therefore, the phosphate group of 2'-GMP cannot locate in the active site but protrudes toward the solvent. This forces 2'-GMP to adopt an anti form, which contrasts with the syn form in the crystal of the intact RNase T1.2'-GMP complex. The inaccessibility of the phosphate group to the active site can account for the lack of the enzymatic activity in CM-RNase T1. One of the carboxyl oxygen atoms of CM-Glu58 forms two hydrogen bonds with the side-chains of Tyr38 and His40. These hydrogen bonds are considered to mainly contribute to the higher thermal stability of CM-RNase T1. Another carboxyl oxygen atoms of CM-Glu58 is situated nearby His40 and Arg77. This may provide additional electrostatic stabilization.

Topics: Binding Sites; Crystallography, X-Ray; Enzyme Stability; Guanosine Monophosphate; Hydrogen Bonding; Models, Molecular; Protein Conformation; Ribonuclease T1

An approach is described for extending free energy calculations to take into account the pH dependence of the relative binding of ligands to an enzyme or other receptor protein. The method is based on the calculation of the free energy difference for a single protonation state via the thermodynamic cycle simulation approach followed by inclusion of all possible protonation states of the enzyme and the inhibitor by use of a macroscopic continuum dielectric (Poisson-Boltzmann) model. A detailed formulation of the combined model is presented. It involves solution of the multiple equilibrium problem and makes use of the calculated pKa values of all titrating groups on both enzyme and ligand. The method is illustrated by calculations of the pH dependence of the differential binding of the inhibitors 2'GMP and 3'GMP to ribonuclease T1. A free energy simulation of the differential binding is made for a given protonation state of the enzyme and inhibitor. Although only qualitative agreement with experiment is obtained, the results provide insights concerning the interactions involved. The pH dependence of the binding is calculated by using the protonation state of the residues from the free energy simulation as the standard state for a Poisson-Boltzmann calculation. Information is obtained concerning the pKa values of the titrating amino acids in the free, 2'GMP and 3'GMP bound enzyme forms of RNase T1 and the difference in the pH dependence of the binding of 2'GMP and 3'GMP to RNase T1. The contributions of different types of interactions (e.g. protein residues versus solvent) to the free energy differences are examined. A free energy simulation of the pKa shift of Glu58 shows that it is important to consider both carboxyl oxygen atoms as possible protonation sites since they may behave very differently in a protein. It is found in the protein that the interactions with the solvent favor the neutral (protonated) state of Glu58. This contrasts sharply with the solution behavior, where the solvent favors the charged state. Analysis of the results shows that the interactions of bound water with other protein residues leads to the observed effect. Comparisons are made with a continuum calculation that uses the charged state employed in the free energy simulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Catalysis; Computer Simulation; Energy Metabolism; Guanosine Monophosphate; Hydrogen-Ion Concentration; Models, Theoretical; Ribonuclease T1

For disordered 2' GMP and 5' GMP the ethidium cation (Etd) was found to form 1:1 and 2:1 Eth:nucleotide complex. For alkali metal solution self-structured 2'GMP and 5'GMP Etd was found to form 1:1 and 2:1 Etd: order nucleotide complex. The best computer fit was obtained for a structured nucleotide stoichiometry of Na4(2'GMP)8. Binding constants for the Etd:disordered 2'GMP complexes were determined to be (1.6 +/- 0.1) x 10(4)M(-1) and 6.3 +/- 0.5 M(-1) at O degrees C and (1.1 +/- 0.2) x 10(4) M(-1) and 18 +/- 11M(-1) for 5'GMP at 5 degrees C for the 1:1 and 1:2 complex, respectively. For the 1:1 and 1:2 2'GMP:Etd species the enthalpies were determined to be -19.8 +/- 1.0 kcal/mole and 0.1 +/- 0.3 kcal/mole, respectively, and the entropies were -53.3 +/- 6.6 eu and 3.9 +/- 1.6 eu, respectively. Binding constants for the Etd: structured 2'GMP complex, assuming a complex with stoichoimetry (Na+)4 (2'GMP)8 for the structured unit, were determined to be (1.9 +/- 0.1) x 10(4)M(-1) and 5.8 +/- 0.6) x 10(2)M(-1), respectively at 0 degrees C.

Topics: Ethidium; Guanosine Monophosphate; Intercalating Agents; Isomerism; Least-Squares Analysis; Magnetic Resonance Spectroscopy; Molecular Structure; Software; Thermodynamics

Proton NMR line broadening methods were used to determine the rates of amino proton exchange for disordered 2'- and 5' - GMP dianions in aqueous solutions containing tetramethylammonium (TMA+) cations. Replacing TMA+ with Na+ does not substantially alter the exchange rates, provided that H-bonded, Na(+)-directed tetramer structures are absent. Activation enthalpies (kcal/mol) and entropies (eu) for 2'-GMP are: delta H not equal to = 18.5 +/- 1.3, delta S not equal to = 9.6 +/- 4.2 for TMA+ salt at pH 8.10, and delta H not equal to = 14.7 +/- 2.6, delta S not equal to = -3.7 +/- 8.0 for the Na+ salt at pH 8.11. Extrapolated values of pseudo first-order rate constants at 25 degrees C are in the range of k = 1-10 sec-1. At suitable concentrations and temperatures, the Na+ salts of both 2'- and 5' - GMP formed stacked and unstacked tetramer units. Relative to the exchange kinetics observed for the disordered nucleotide, the exchange process in the tetramer units was catalyzed in half the amino protons and inhibited in the other half. The catalytic process (k > 10(3) sec-1) has been attributed to amino protons not involved in interbase H-bonding, where as the inhibited process (k < 10(-1) sec-1) was assigned to those protons which do form such bonds. The structure-catalyzed process in both the stacked and unstacked tetramers was manifested by a loss of NMR amino proton intensity due to weighted time-averaging with the resonance for bulk water. A bridging water molecule between an amino proton and a phosphate on an adjacent nucleotide in the tetramer unit may provide a mechanistic pathway for the structure-catalyzed process.

Topics: Guanosine Monophosphate; Hydrogen Bonding; Kinetics; Magnetic Resonance Spectroscopy; Molecular Structure; Protons; Quaternary Ammonium Compounds; Sodium; Solutions; Water

The results of 1-nanosecond molecular dynamics simulations of the enzyme ribonuclease T1 and its 2'GMP complex in water are presented. A classification of the angular reorientations of the backbone amide groups is achieved via a transformation of NH-vector trajectories into several coordinate frames, thus unravelling contributions of NH-bond librations and backbone dihedral angle fluctuations. The former turned out to be similar for all amides, as characterized by correlation times of librational motions in a subpicosecond scale, angular amplitudes of about 10-12 degrees for out-of-peptide-plane displacements of the NH-bond and 3-5 degrees for the in-plane displacements, whereas the contributions of much slower backbone dihedral angle fluctuations strongly depend on the secondary structure. Correlation functions relevant for NMR were obtained and analyzed utilizing the 'model-free' approach (Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570; Clore et al., (1990) J. Am. Chem. Soc. 112, 4989-4991). The dependence of the amplitude of local motion on the residue location in the backbone is in good agreement with the results of NMR relaxation measurements and X-ray data. The protein dynamics is characterized by a highly restricted local motion of those parts of the backbone with defined secondary structure as well as by a high flexibility in loop regions. The comparison of results derived from different periods of the trajectory (of 50 ps and 1 ns duration, 1000 points sampled) reveals a dependence of the observed dynamic picture on the characteristic time scale of the experimental method used. Comparison of the MD data for the free and liganded enzyme clearly indicates a restriction of the mobility within certain regions of the backbone upon inhibitor binding.

Topics: Computer Simulation; Crystallography, X-Ray; Guanosine Monophosphate; Isomerism; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Structure; Protein Structure, Secondary; Ribonuclease T1; Water

The structure of the complex of ribonuclease from Streptomyces aureofaciens (RNase Sa) with exo guanosine 2',3'-cyclophosphorothioate has been refined against 0.2-nm resolution synchrotron data using, as a starting model, coordinates from the RNase Sa: 2'-GMP complex. The refinement was based on all data over 1.0-0.2 nm and converged to a crystallographic R factor of 11.9%. This is the first structure of a microbial ribonuclease complexed with a 2',3'-cyclophosphorothioate, which is a thio analogue of the intermediate of the two-step reaction. However, exo guanosine 2',3'-cyclophosphorothioate is bound in a non-functional mode and is not hydrolysed. This structure therefore does not provide direct evidence on the identity of the amino acid residues responsible for catalytic cleavage of the substrate. However, based on present and previous results, a plausible model is proposed for the complex of the cyclic intermediate which acts as substrate for the second step of the catalysis.

Topics: Binding Sites; Crystallization; Cyclic GMP; Guanosine Monophosphate; Hydrolysis; Ribonucleases; Streptomyces aureofaciens; Temperature; Thionucleotides; X-Ray Diffraction

The binding of the mononucleotide inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to genetically engineered ribonuclease T1 has been investigated by conventional inhibition kinetics, fluorimetric titrations, molecular modeling, and fast relaxation techniques. The fluorimetric titrations in conjunction with molecular modeling revealed that apart from the already known primary binding site, three to four additional sites are present on the enzyme's surface. The association constants obtained from the fluorimetric titrations and the temperature jump experiments range between 3.1 x 10(6) M-1 and 4.3 x 10(6) M-1, indicating that the binding of the mononucleotides to the specific binding site of ribonuclease T1 is at least one order of magnitude tighter than has been anticipated so far. The kinetics of binding are nearly diffusion controlled with a kon determined for 2'-GMP and 3'-GMP, as (5.0 +/- 0.5 x 10(9) and 6.1 +/- 0.5 x 10(9) M-1, s-1 and koff as 1.2 +/- 0.2 x 10(3) and 2.0 +/- 0.3 x 10(3) s-1, respectively. Molecular modeling studies indicate that all three nucleotides are able to bind via their phosphate group to a positively charged array of surface amino acids including His27, His40, Lys41, and most probably Lys25 without obvious stereochemical hindrance. We propose that RNA wraps around RNase T1 in a similar fashion via phosphate binding when enzymatic hydrolysis occurs.

Topics: Binding Sites; Computer Simulation; Guanosine Monophosphate; Kinetics; Models, Molecular; Ribonuclease T1; Spectrometry, Fluorescence; Thermodynamics

Phosphorescence and ODMR measurements have been made on ribonuclease T1 (RNase T1), the mutated enzyme RNase T1 (Y45W), and their complexes with 2'GMP and 2'AMP. It is not possible to observe the phosphorescence of Trp45 in RNase T1 (Y45W). Only that of the naturally occurring Trp59 is seen. The binding of 2'GMP to wild-type RNase T1 produces only a minor red shift in the phosphorescence and no change in the ODMR spectrum of Trp59. However, a new tryptophan 0,0-band is found 8.2 nm to the red of the Trp59 0,0-band in the 2'GMP complex of the mutated RNase T1 (Y45W). Wavelength-selected ODMR measurements reveal that the red-shifted emission induced by 2'GMP binding, assigned to Trp45, occurs from a residue with significantly different zero-field splittings than those of Trp59, a buried residue subject to local polar interactions. The phosphorescence red shift and the zero-field splitting parameters demonstrate that Trp45 is located in a polarizable environment in the 2'GMP complex. In contrast with 2'GMP, binding of 2'AMP to RNase T1 (Y45W) induces no observable phosphorescence emission from Trp45, but leads only to a minor red shift in the phosphorescence origin of Trp59 in both the mutated and wild-type enzyme. The lack of resolved phosphorescence emission from Trp45 in RNase T1 (Y45W) implies that the emission of this residue is quenched in the uncomplexed enzyme. We conclude that local conformational changes that occur upon binding 2'GMP remove quenching residues from the vicinity of Trp45, restoring its luminescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Monophosphate; Amino Acid Sequence; Binding Sites; Guanosine Monophosphate; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Binding; Protein Conformation; Recombinant Proteins; Ribonuclease T1; X-Ray Diffraction

On the basis of molecular dynamics and free-energy perturbation approaches, the Glu46Gln (E46Q) mutation in the guanine-specific ribonuclease T1 (RNase T1) was predicted to render the enzyme specific for adenine. The E46Q mutant was genetically engineered and characterized biochemically and crystallographically by investigating the structures of its two complexes with 2'AMP and 2'GMP. The ribonuclease E46Q mutant is nearly inactive towards dinucleoside phosphate substrates but shows 17% residual activity towards RNA. It binds 2'AMP and 2'GMP equally well with dissociation constants of 49 microM and 37 microM, in contrast to the wild-type enzyme, which strongly discriminates between these two nucleotides, yielding dissociation constants of 36 microM and 0.6 microM. These data suggest that the E46Q mutant binds the nucleotides not to the specific recognition site but to the subsite at His92. This was confirmed by the crystal structures, which also showed that the Gln46 amide is hydrogen bonded to the Phe100 N and O atoms, and tightly anchored in this position. This interaction may either have locked the guanine recognition site so that 2'AMP and 2'GMP are unable to insert, or the contribution to guanine recognition of Glu46 is so important that the E46Q mutant is unable to function in recognition of either guanine and adenine.

Topics: Adenosine Monophosphate; Base Sequence; Binding Sites; Exoribonucleases; Glutamates; Glutamic Acid; Glutamine; Guanosine Monophosphate; Isomerism; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; X-Ray Diffraction

We present free energy perturbation calculations on the complexes of Glu46----Ala46 (E46A) and Glu46----Gln46 (E46Q) mutants of ribonuclease T1 (RNaseT1) with inhibitors 2'-guanosine monophosphate (GMP) and 2'-adenosine monophosphate (AMP) by a thermodynamic perturbation method implemented with molecular dynamics (MD). Using the available crystal structure of the RNaseT1-GMP complex, the structures of E46A-GMP and E46Q-GMP were model built and equilibrated with MD simulations. The structures of E46A-AMP and E46Q-AMP were obtained as a final structure of the GMP----AMP perturbation calculation respectively. The calculated difference in the free energy of binding (delta delta Gbind) was 0.31 kcal/mol for the E46A system and -1.04 kcal/mol for the E46Q system. The resultant free energies are much smaller than the experimental and calculated value of approximately 3 kcal/mol for the native RNaseT1, which suggests that both mutants have greater relative adenine affinities than native RNaseT1. Especially E46Q is calculated to have a larger affinity for adenine than guanine, as we suggested previously from the calculation on the native RNaseT1. Thus, the molecular dynamics/free energy perturbation method may be helpful in protein engineering, directed toward increasing or changing the substrate specificity of enzymes.

Topics: Adenosine Monophosphate; Binding Sites; Energy Transfer; Guanosine Monophosphate; Hydrogen Bonding; Mathematics; Models, Molecular; Molecular Conformation; Mutagenesis, Site-Directed; Ribonuclease T1; Substrate Specificity; Thermodynamics; X-Ray Diffraction

The structure of RNase F1 in aqueous solution has been studied by Raman spectroscopy and compared with that of a homologous enzyme, RNase T1. RNase F1 contains less beta-sheet and alpha-helical structure and more irregular structure than RNase T1. The strength of hydrogen bonding is weak in the beta-sheet and strong in the alpha-helix compared to that of RNase T1. Two disulfide bridges take the gauche-gauche and gauche-trans conformations, respectively. The overall hydrogen bonding of nine Tyr side chains in RNase F1 is very similar to that in RNase T1. Both of two His residues have pKa values around 8.2, which are close to those of the His residues in the active site of RNase T1. Upon binding of 2'-GMP, the hydrogen bonding of some Tyr side chains changes to a more proton-donating state. 2'-GMP is strongly hydrogen bonded with the enzyme at N7 of the guanine ring and takes the C3' endo-syn conformation. The binding mode of the inhibitor is identical to that found for RNase T1. In spite of significant differences in secondary structure, the molecular architecture of the active site seems to be highly conserved.

Topics: Amino Acid Sequence; Binding, Competitive; Cations; Cysteine; Disulfides; Fusarium; Guanosine Monophosphate; Histidine; Hydrogen Bonding; Protein Conformation; Ribonuclease T1; Spectrum Analysis, Raman; Stereoisomerism; Tyrosine

Complex of a mutant ribonuclease T1 (Y45W) with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined by X-ray diffraction at 1.7 A resolution. The 2'AMP molecule locates at a new base-binding site which is remote from the guanine-recognition site, where 2'GMP was found to be bound. The nucleotide adopts the anti conformation of the glycosidic bond and C3'-exo sugar pucker. There exists a single hydrogen bond between the adenine base and the enzyme, and, therefore, the site found is apparently a non-specific binding site. The results indicate that the binding of 2'AMP to the guanine-recognition site is weaker than that to the new binding site.

Topics: Adenosine Monophosphate; Amino Acid Sequence; Aspergillus oryzae; Binding Sites; Crystallography; Guanosine Monophosphate; Hydrogen Bonding; Isomerism; Models, Molecular; Molecular Structure; Mutation; Recombinant Proteins; Ribonuclease T1; Structure-Activity Relationship; X-Ray Diffraction

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a specific inhibitor, 2'GMP, has been determined by X-ray diffraction and refined at 1.9 A resolution to a conventional R-factor of 0.164. The mode of recognition of the guanine base by the enzyme is similar to that found for the wild-type ribonuclease T1 complexed with 2'GMP. The binding of the guanine base is clearly enhanced by maximum overlapping of the indole ring of Trp45 and the base. The glycosyl torsion angle of the inhibitor is in the syn conformation and the sugar exhibits a C3'-endo type pucker, which differs from that observed in the crystal of the complex between the wild-type ribonuclease T1 and 2'GMP. Analysis of 500-MHZ NMR spectra has also indicated that the 2'GMP molecule as bound to the mutant enzyme in solution exhibits a C3'-endo type pucker, similar to that bound to the wild-type enzyme in solution [Inagaki, Shimada, & Miyazawa (1985) Biochemistry 24, 1013-1020].

Topics: Carbohydrate Conformation; Escherichia coli; Guanosine Monophosphate; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Mutation; Protein Binding; Protein Conformation; Ribonuclease T1; Solutions; Stereoisomerism; X-Ray Diffraction

Hydrophobic effects on binding of ribonuclease T1 to guanine bases of several ribonucleotides have been proved by mutating a hydrophobic residue at the recognition site and by measuring the effect on binding. Mutation of a hydrophobic surface residue to a more hydrophobic residue (Tyr45----Trp) enhances the binding to ribonucleotides, including mononucleotide inhibitor and product, and a synthetic substrate-analog trinucleotide as well as the binding to dinucleotide substrates and RNA. Enhancements on binding to non-substrate ribonucleotides by the mutation have been observed with free energy changes ranging from -2.2 to -3.9 kJ/mol. These changes are in good agreement with that of substrate binding, -2.3 kJ/mol, which is calculated from Michaelis constants obtained from kinetic studies. It is shown, by comparing the observed and calculated changes in binding free energy with differences in the observed transfer free energy changes of the amino acid side chains from organic solvents to water, that the enhancement observed on guanine binding comes from the difference in the hydrophobic effects of the side chains of tyrosine and tryptophan. Furthermore, a linear relationship between nucleolytic activities and hydrophobicity of the residues (Ala, Phe, Tyr, Trp) at position 45 is observed. The mutation could not change substantially the base specificity of RNase T1, which exhibits a prime requirement for guanine bases of substrates.

Topics: Binding Sites; Guanosine Monophosphate; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Oligoribonucleotides; Protein Conformation; Ribonuclease T1; Ribonucleotides; RNA; Surface Properties; Tryptophan

Fluorescence titrations and temperature-jump relaxation experiments were performed as a function of temperature on ribonuclease T1 with the inhibitors 2'GMP and 3'GMP to obtain information on the energetics and molecular events controlling the binding of those inhibitors. Results from the titration and temperature-jump experiments were in agreement concerning the equilibrium constant. The larger equilibrium constant for 2'GMP is enthalpic in origin and is due to both a higher on rate and a lower off rate as compared to 3'GMP. On rates for both inhibitors appear to be below the diffusion controlled limit, apparently due to conformational changes in the portion of the active site responsible for recognition of the guanine base. Comparison of the measured enthalpic and entropic terms associated with the equilibrium constant determined from the fluorescence titrations are in disagreement with those calculated from the on and off rates indicating the presence of an induced conformational change in the 2'GMP-enzyme complex. This second conformational change appears to be due to additional interactions between 2'GMP and the catalytic portion of the active site, which may also be responsible for the differences in the binding constant, the on rate and the off rate between 2'GMP and 3'GMP.

Topics: Binding Sites; Chemical Phenomena; Chemistry, Physical; Diffusion; Guanosine Monophosphate; Hydrogen-Ion Concentration; Protein Conformation; Ribonuclease T1; Spectrometry, Fluorescence; Temperature; Thermodynamics

We present a calculation of the relative changes in binding free energy between the complex of ribonuclease T1 (RNase Tr) with its inhibitor 2'-guanosine monophosphate (2'GMP) and that of RNase T1-2'-adenosine monophosphate (2'AMP) by means of a thermodynamic perturbation method implemented with molecular dynamics. Using the available crystal structure of the RNase T1-2'GMP complex, the structure of the RNase T1-2'AMP complex was obtained as a final structure of the perturbation calculation. The calculated difference in the free energy of binding (delta delta Gbind) was 2.76 kcal/mol. This compares well with the experimental value of 3.07 kcal/mol. The encouraging agreement in delta delta Gbind suggests that the interactions of inhibitors with the enzyme are reasonably represented. Energy component analyses of the two complexes reveal that the active site of RNase T1 electrostatically stabilizes the binding of 2'GMP more than that of 2'AMP by 44 kcal/mol, while the van der Waals' interactions are similar in the two complexes. The analyses suggest that the mutation from Glu46 to Gln may lead to a preference of RNase T1 for adenine in contrast to the guanine preference of the wild-type enzyme. Although the molecular dynamics equilibration moves the atoms of the RNase T1-2'GMP system about 0.9 A from their X-ray positions and the mutation of the G to A in the active site increases the deviation from the X-ray structure, the mutation of the A back to G reduces the deviation. This and the agreement found for delta delta Gbind suggest that the molecular dynamics/free energy perturbation method will be useful for both energetic and structural analysis of protein-ligand interactions.

Topics: Adenosine Monophosphate; Amino Acid Sequence; Aspergillus oryzae; Computer Graphics; Exoribonucleases; Guanine Nucleotides; Guanosine Monophosphate; Hydrogen Bonding; Models, Molecular; Molecular Sequence Data; Ribonucleases; Sequence Homology, Nucleic Acid; Software; Thermodynamics

The enzyme ribonuclease T1 (RNase T1) isolated from Aspergillus oryzae was cocrystallized with the specific inhibitor guanylyl-2',5'-guanosine (2',5'-GpG) and the structure refined by the stereochemically restrained least-squares refinement method to a crystallographic R-factor of 14.9% for X-ray data above 3 sigma in the resolution range 6 to 1.8 A. The refined model consists of 781 protein atoms, 43 inhibitor atoms in a major site and 29 inhibitor atoms in a minor site, 107 water oxygen atoms, and a metal site assigned as Ca. At the end of the refinement, the orientation of His, Asn and Gln side-chains was reinterpreted on the basis of two-dimensional nuclear magnetic resonance data. The crystal packing and enzyme conformation of the RNase T1/2',5'-GpG complex and of the near-isomorphous RNase T1/2'-GMP complex are comparable. The root-mean-square deviation is 0.73 A between equivalent protein atoms. Differences in the unit cell dimensions are mainly due to the bound inhibitor. The 5'-terminal guanine of 2',5'-GpG binds to RNase T1 in much the same way as in the 2'-GMP complex. In contrast, the hydrogen bonds between the catalytic center and the phosphate group are different and the 3'-terminal guanine forms no hydrogen bonds with the enzyme. This poor binding is reflected in a 2-fold disorder of 2',5'-GpG (except the 5'-terminal guanine), which originates from differences in the pucker of the 5'-terminal ribose. The pucker is C2'-exo for the major site (2/3 occupancy) and C1'-endo for the minor site (1/3 occupancy). The orientation of the major site is stabilized through stacking interactions between the 3'-terminal guanine and His92, an amino acid necessary for catalysis. This might explain the high inhibition rate observed for 2',5'-GpG, which exceeds that of all other inhibitors of type 2',5'-GpN. On the basis of distance criteria, one solvent peak in the electron density was identified as metal ion, probably Ca2+. The ion is co-ordinated by the two Asp15 carboxylate oxygen atoms and by six water molecules. The co-ordination polyhedron displays approximate 4m2 symmetry.

Topics: Aspergillus; Binding Sites; Calcium; Dinucleoside Phosphates; Exoribonucleases; Guanosine Monophosphate; Hydrogen Bonding; Macromolecular Substances; Models, Molecular; Molecular Conformation; Sodium; X-Ray Diffraction

The three-dimensional X-ray structure of the RNase T1[EC 3.1.27.3]-2'GMP complex crystallized at low pH value (4.0) was determined, and refined to 1.9 A resolution to give a final R value of 0.203. The refined model includes 781 protein atoms, 24 inhibitor atoms, and 43 solvent molecules. The imidazole rings of His27 and His40 interact with the carboxyl side chains of Glu82 and Glu58, respectively, whereas that of His92 is in contact with the main chain carbonyl oxygen of Ala75. In the complex, the ribose ring of the 2'GMP molecule adopts a C2'-endo puckering, and the exocyclic conformation is gauche(-)-gauche(+). The glycosyl torsion angle is in the syn range with an intramolecular hydrogen bond between N3 and O5', and the 2'-phosphate orientation is trans-gauche(-). The guanine base of the inhibitor is tightly bound to the base recognition site with five hydrogen bonds (N1--Glu46O epsilon 2, N2---Asn98O,O6---Asn44N, and N7 ---Asn43N delta 2/Asn43N) and is sandwiched between the phenolic ring portions of Tyr42 and Tyr45 by stacking interactions. The 2'-phosphate group interacts with Arg77N eta 2, Glu58O episilon 2, and Tyr 38O eta but not with any of the histidine residues. Arg77N eta 2 also interacts with Tyr38O eta. There is no interaction between the ribose moiety of the inhibitor and the enzyme.

Topics: Crystallization; Endoribonucleases; Guanine Nucleotides; Guanosine Monophosphate; Hydrogen Bonding; Hydrogen-Ion Concentration; Macromolecular Substances; Models, Molecular; Ribonuclease T1; Solvents; X-Ray Diffraction

The complex formed between the enzyme ribonuclease T1 (EC 3.1.27.3) and its specific inhibitor 2'-guanylic acid (2'-GMP) has been refined to R = 0.180 using x-ray diffraction data to 1.9-A resolution. The protein molecule displays a compact fold; a 4.5 turn alpha-helix packed over an antiparallel beta-pleated sheet shields most of the hydrophobic interior of the protein against the solvent. The extended pleated sheet structure of ribonuclease T1 is composed of three long and four short strands building up a two-stranded minor beta-sheet near the amino terminus and a five-stranded major sheet in the interior of the protein molecule. In the complex with ribonuclease T1, the inhibitor 2'-guanylic acid adopts the syn-conformation and C2'-endo sugar pucker. Binding of the nucleotide is mainly achieved through amino acid residues 38-46 of the protein. The catalytically active amino acid residues of ribonuclease T1 (His40, Glu58, Arg77, and His92) are located within the major beta-sheet which, as evident from the analysis of atomic temperature factors, provides an environment of minimal local mobility. The geometry of the active site is consistent with a mechanism for phosphodiester hydrolysis where, in the transesterification step, His40 and/or Glu58 act as a general base toward the ribose 2'-hydroxyl group and His92, as a general acid, donates a proton to the leaving 5'-hydroxyl group.

Topics: Endoribonucleases; Guanosine Monophosphate; Hydrogen Bonding; Models, Molecular; Protein Conformation; Ribonuclease T1

RNase T1 is folded into an alpha-helix of 4.5 turns, covered by a four-strand antiparallel beta-sheet. Specific recognition of 2'-guanylic acid arises from hydrogen bonding between main chain peptide groups and the O-6 and N-1-H of guanine, as well as from stacking of Tyr 45 on guanine. At the active site, Glu 58, His 92 and Arg 77 are involved in phosphodiester hydrolysis.

Topics: Amino Acid Sequence; Catalysis; Guanine Nucleotides; Guanosine Monophosphate; Models, Molecular; Protein Binding; Protein Conformation; Ribonuclease T1; Ribonucleases; Structure-Activity Relationship; Substrate Specificity; X-Ray Diffraction

Ribonuclease T1 was crystallized under various conditions. Form I crystals were produced by microdialysis against 53% (v/v) 2-methyl-2,4-pentanediol in 0.01 M sodium acetate, 0.05% 2'-guanylic acid (2'GMP) and 0.02% NaN3 (pH 6.2-7.2). These crystals are tetragonal, space group P41212 and contain two molecules per asymmetric unit; cell dimensions are a = b = 5.86 nm, c = 13.28 nm. Form IIa and form IIb crystals were obtained by microdialysis from a buffer of 0.01-0.05 M sodium acetate, 0.25-0.5% 2'GMP, 0.02% NaN3 and 2-5 mM calcium acetate (pH 4.0-4.4) in the presence of 50-75% (v/v) 2-methyl-2,4-pentanediol. These crystals are orthorhombic, space group P212121, and contain one molecule per asymmetric unit; cell dimensions are a = 4.66 nm, b = 5.02 nm, c = 4.04 nm (form I) and alpha = 4.44 nm, b = 5.00 nm, c = 4.03 nm (form II). Using high-performance liquid chromatography, it could be shown for all crystal forms that 2'-GMP is bound in the crystals. The molecular ratio between RNase T1 and 2'GMP was 0.9 for form II crystals and thus agreed with a 1:1 enzyme-nucleotide complex. Heavy-atom derivatives were produced with lead acetate for form IIa crystals and with uranyl acetate for from IIb crystals. Three-dimensional X-ray analysis of the RNase-T1 x 2'GMP complex is under way.

Topics: Aspergillus oryzae; Binding Sites; Crystallization; Guanine Nucleotides; Guanosine Monophosphate; Protein Binding; Protein Conformation; Ribonuclease T1; Ribonucleases; X-Ray Diffraction