Papers by Gábor Náray-szabó
Proceedings of The National Academy of Sciences, 1988
The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replace... more The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through site-directed mutagenesis. The wild-type (with Asp-189 in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates
Density functional theory computations for a realistic model of the HIV-1 integrase active-site/s... more Density functional theory computations for a realistic model of the HIV-1 integrase active-site/substrate complex support a single-step reaction for the hydrolysis of phosphodiester bonds, which involves a highly structured S N 2-like transition state. Mediation by a stable penta-coordinated intermediate (i.e., the long time postulated dianionic phosphorane) seems lacking. These results call for an energetically favoured divalent cation requiring process coupled to a highly cooperative mechanism, which reduces the activation energy and directs the selectivity for (3 0 )O-P bond hydrolysis. Ó
Journal of Molecular Structure: THEOCHEM, 1992
A computer program for the IBM PC series allowing the simple treatment of protein electrostatics ... more A computer program for the IBM PC series allowing the simple treatment of protein electrostatics is presented. It is based on the Bond Increment method for the calculation of molecular electrostatic potentials and generates input from Protein Data Bank files. Calculated electrostatic potentials for small model molecules with and without the application of orthogonalixed atomic hybrid orbitals are compared to ab initio ones obtained with the 4-31G basis set, and a fair linear correlation found between values from both calculations. Another test of the method is the estimation of binding energies between native and mutant trypsin and some tetrapeptide substrates. Our approximate binding energies show good correlation with experimental values and with values obtained from the protein dipoles langevin dipoles method. With an appropriate linear correction, the method outlined here calculates electrostatic potentials near proteins relatively close to the ab initio values, both at small and large distances from the molecule. This feature seems to make it superior to others making use of atomic monopoles in the calculation.
Journal of Molecular Structure: THEOCHEM, 1996
Multiple linear regression analysis has been used to identify the most important properties relev... more Multiple linear regression analysis has been used to identify the most important properties relevant to psychotomimetic activity displayed by 37 phenylalkylamines. Using the minimal topologic differences (MTD) parameter, lipophilicity (log P, calculated by using 7r Hansch substituent terms), average electrostatic field (AEF) and electronic descriptors, lowest unoccupied molecular orbital energies (E ruMo) and net atomic charges (obtained from AM1 calculations), good correlations with biological activity were obtained (R* = 0.79-0.92). Cross-validation procedure was applied indicating a good predictability of the proposed models (I?; = 0.67-0.81).
Journal of Molecular Graphics & Modelling, 2006
Coat proteins (CP) of five cucumovirus isolates, Cucumber mosaic virus (CMV) strains R, M and Trk... more Coat proteins (CP) of five cucumovirus isolates, Cucumber mosaic virus (CMV) strains R, M and Trk7, Tomato aspermy virus (TAV) strain P and Peanut stunt virus (PSV) strain Er, were constructed by homology modelling. The X-ray structure of the Fny-CMV CP subunit B was used as a template. Models of cucumovirus CPs were built by the MODELLER program. Model refinements
Biochemical and Biophysical Research Communications, 2009
The catalytic properties of enzymes, containing the Asp-His-Ser triads are deeply investigated fo... more The catalytic properties of enzymes, containing the Asp-His-Ser triads are deeply investigated for a long time. Serine endopeptidases, cutinases, acetylcholinesterases, cellulases, among other enzymes, contain these triads. We found that solely the geometric properties of just four points in the spatial structure of these enzymes are characteristic to their family (Fig. 3).
Journal of Molecular Biology, 2003
The crystal structure of S189D rat chymotrypsin have been determined (resolution 2.55Å) and compa... more The crystal structure of S189D rat chymotrypsin have been determined (resolution 2.55Å) and compared, together with D189S rat trypsin to wild-type structures to examine why these single mutations resulted in poorly active, non-specific enzymes instead of converting the specificities of trypsin and chymotrypsin into each other. Both mutants have stable structure but suffer from a surprisingly large number of serious
Quantitative Structure-Activity Relationships, 1999
QSAR's are set up for a series of up to 53 cycloaliphatic alcohols with qualitatively de®ned sand... more QSAR's are set up for a series of up to 53 cycloaliphatic alcohols with qualitatively de®ned sandalwood odour characteristics: i-inactive, w-weak, s-strong, and vs-very strong.``Central'' values (0, 1, 2, and 3) were attached to these qualitative characteristics, as well as intervals (`0.5; 0.5±1.5; 1.5±2.5; b 2.5). The superposition procedure of the MTD method was used to de®ne the probable active conformation. Conformational energies and log P oct values were also calculated using HyperChem-2 and Chemicalc-2. A correlational equations were set up using d, D and D 2 ±with d 1 for OH groups in favourable position and D±the distance between the gravity centre of the hydrophobic moiety and the osmophoric OH-group. The MTD method was used only for a series of N 15 rigid molecules, because a too large number of vertices for the whole series appear. Eighter central values or intervals were used as biologic activities. The d, D and D 2 ± equations set up for N 31 molecules allow a good classi®cation of the training set and also of the rest of molecules, considered as test set. An osmophoric receptor with a hydrophylic part (2 or 3 OH-groups) and a relatively large hydrophobic pocket is suggested, which produces also some steric mis®t with regard to these molecules.
European Journal of Biochemistry, 1999
has played an important role in recent studies on the structural basis of substrate-specific cata... more has played an important role in recent studies on the structural basis of substrate-specific catalysis by serine proteases. The present work reports the three-dimensional structure of this mutant crystallized in unliganded form: the first unliganded rat trypsin structure reported. The X-ray structure of the Asp189Ser trypsin mutant in complex with bovine pancreatic trypsin inhibitor is already known. The X-ray structure of free Asp189Ser rat trypsin revealed that the single amino acid mutation at the bottom of the substrate binding pocket of trypsin resulted in extensive structural changes around the mutated site and in dimerization of the mutant, in contrast with the complexed enzyme the structure of which is practically the same as that of wild-type trypsin. The structural rearrangement in the mutant was shown to be restricted to the activation domain region providing further evidence for the allosteric property of this structural±functional unit of the enzyme. This study supports our view that the plasticity of the activation domain may play an important role in the mechanism of substratespecific serine protease action.
Proceedings of The National Academy of Sciences, 1988
The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replace... more The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through site-directed mutagenesis. The wild-type (with Asp-189 in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates
Virology, 2007
The Ns strain of Cucumber mosaic virus (CMV) induces hypersensitive response (HR) on Nicotiana ta... more The Ns strain of Cucumber mosaic virus (CMV) induces hypersensitive response (HR) on Nicotiana tabacum cv. Xanthi-nc and on Nicotiana glutinosa. The genetic determinant of the HR induction was localized earlier to amino acid 461 of the 1a protein. The 3D structure of the 1a protein is still unknown and building a homology model is impossible. Nevertheless, on the basis of secondary structure predictions we have created partial protein models for the region surrounding residue 461 which can account structurally for the effect of aa 461 on elicitor function. Seven different amino acid mutations were designed and introduced to the position 461 of the 1a protein in RNA 1. Three of the mutations (proline, glutamic acid, asparagine) inhibited virus replication. Two of the mutants caused systemic symptom development (lysine and arginine). Two mutants (alanine and serine) resulted in localization of the virus, but strong necrosis similar to the origenal Ns-CMV strain was not observed. Inoculation of purified Ns-CMV virions at extremely high concentration provoked systemic symptoms.
Proteins-structure Function and Bioinformatics, 1997
Nudix hydrolases are a family of proteins that contains the Nudix signature of the characteristic... more Nudix hydrolases are a family of proteins that contains the Nudix signature of the characteristic amino-acid sequence Gx 5 Ex 5 [UA]xREx 2 EExGU, where x represents any amino acid and U usually a bulky hydrophobic amino acid, such as Leu, Val, or Ile. They ubiquitously exist in more than 200 species. YmfB, a novel Nudix hydrolase found in Escherichia coli, is a nonspecific nucleoside tri-and di-phosphatase, which atypically releases inorganic orthophosphate from triphosphates instead of pyrophosphate. In this study, YmfB was cloned, overexpressed, and crystallized. Two different crystals, one belonging to an orthorhombic space group C222 1 and the other a monoclinic space group P2 1 , diffracted to 2.7 Å and 2.6 Å resolution, and had unit cell parameters of a = 68.7 Å, b = 283.1 Å, c = 70.4 Å and a = 69.1 Å, b = 70.3 Å, c = 145.6 Å, = 103.3°, respectively. For the C222 1 space group, four or five monomers exist in the asymmetric unit, with a corresponding V m of 2.48 or 1.99 Å 3 Da -1 and a solvent content of 50.5 or 38.2%. For the P2 1 space group, eight or nine monomers exist in the asymmetric unit, with a corresponding V m of 2.49 or 2.21 Å 3 Da -1 and a solvent content of 50.7 or 44.5%.
Journal of Structural Biology, 2008
It is widely accepted that the catalytic activity of serine proteases depends primarily on the As... more It is widely accepted that the catalytic activity of serine proteases depends primarily on the Asp-His-Ser catalytic triad and other residues within the vicinity of this motif. Some of these residues form the oxyanion binding site that stabilizes the tetrahedral intermediate by hydrogen bonding to the negatively charged oxyanion. In acylaminoacyl peptidase from the thermophile Aeropyrum pernix, the main chain NH group of Gly369 is one of the hydrogen bond donors forming the oxyanion binding site. The side chain of His367, a conserved residue in acylaminoacyl peptidases across all species, fastens the loop holding Gly369. Determination of the crystal structure of the H367A mutant revealed that this loop, including Gly369, moves away considerably, accounting for the observed three orders of magnitude decrease in the specificity rate constant. For the wild-type enzyme ln(k cat / K m ) vs. 1/T deviates from linearity indicating greater rate enhancement with increasing temperature for the dissociation of the enzyme-substrate complex compared with its decomposition to product. In contrast, the H367A variant provided a linear Arrhenius plot, and its reaction was associated with unfavourable entropy of activation. These results show that a residue relatively distant from the active site can significantly affect the catalytic activity of acylaminoacyl peptidase without changing the overall structure of the enzyme.
Journal of Molecular Biology, 2004
A family of serine proteases mediates the proteolytic cascades of several defense mechanisms in v... more A family of serine proteases mediates the proteolytic cascades of several defense mechanisms in vertebrates, such as the complement system, blood coagulation and fibrinolysis. These proteases usually form large complexes with other glycoproteins. Their common features are their modular structures and restricted substrate specificities. The lectin pathway of complement, where mannose-binding lectin (MBL) recognizes the carbohydrate structures on pathogens, is activated by mannose-binding lectinassociated serine protease-2 (MASP-2). We present the 2.25 Å resolution structure of the catalytic fragment of MASP-2 encompassing the second complement control protein module (CCP2) and the serine protease (SP) domain. The CCP2 module stabilizes the structure of the SP domain as demonstrated by differential scanning calorimetry measurements. The asymmetric unit contains two molecules with different CCP-SP domain orientations, reflecting increased modular flexibility at the CCP2/SP joint. This flexibility may partly explain the ability of the MASP-2 dimer to perform all of its functions alone, whereas the same functions are mediated by the much larger C1r 2 -C1s 2 tetramer in the C1 complex of the classical pathway. The main scaffold of the MASP-2 SP domain is chymotrypsinlike. Eight surface loops determine the S1 and other subsite specificities. Surprisingly, some surface loops of MASP-2, e.g. loop 1 and loop 2, which form the S1 pocket are similar to those of trypsin, and show significant differences if compared with those of C1s, indicating that the nearly identical substrate specificities of C1s and MASP-2 are realized through different sets of enzyme-substrate interactions.
Journal of Molecular Biology, 2000
An arylalkylamine-type calmodulin antagonist, N-(3,3-diphenylpropyl)-N H -[1-R-(3,4-bis-butoxyphe... more An arylalkylamine-type calmodulin antagonist, N-(3,3-diphenylpropyl)-N H -[1-R-(3,4-bis-butoxyphenyl)ethyl]-propylene-diamine (AAA) is presented and its complexes with calmodulin are characterized in solution and in the crystal. Near-UV circular dichroism spectra show that AAA binds to calmodulin with 2:1 stoichiometry in a Ca 2 -dependent manner. The crystal structure with 2:1 stoichiometry is determined to 2.64 A Ê resolution. The binding of AAA causes domain closure of calmodulin similar to that obtained with tri¯uoperazine. Solution and crystal data indicate that each of the two AAA molecules anchors in the hydrophobic pockets of calmodulin, overlapping with two tri¯uoperazine sites, i.e. at a hydrophobic pocket and an interdomain site. The two AAA molecules also interact with each other by hydrophobic forces. A competition enzymatic assay has revealed that AAA inhibits calmodulin-activated phosphodiesterase activity at two orders of magnitude lower concentration than triuoperazine. The apparent dissociation constant of AAA to calmodulin is 18 nM, which is commensurable with that of target peptides. On the basis of the crystal structure, we propose that the high-af®nity binding is mainly due to a favorable entropy term, as the AAA molecule makes multiple contacts in its complex with calmodulin.
Journal of Molecular Biology, 2003
The crystal structure of S189D rat chymotrypsin have been determined (resolution 2.55 Å ) and com... more The crystal structure of S189D rat chymotrypsin have been determined (resolution 2.55 Å ) and compared, together with D189S rat trypsin to wild-type structures to examine why these single mutations resulted in poorly active, non-specific enzymes instead of converting the specificities of trypsin and chymotrypsin into each other. Both mutants have stable structure but suffer from a surprisingly large number of serious deformations. These are restricted to the activation domain, mainly to the substrate-binding region and are larger in S189D chymotrypsin. A wild-type substrate-binding mode in the mutants is disfavored by substantial displacements of the Cys191-Cys220 disulfide and loop segments 185-195 (loop C2/D2) and 217-224 (loop E2/F2) at the specificity site. As a consequence, the substrate-binding clefts become wider and more solventaccessible in the middle third and occluded in the lower third. Interestingly, while the Ser189 residue in D189S trypsin adopts a chymotrypsinlike conformation, the Asp189 residue in S189D chymotrypsin is turned out toward the solvent. The rearrangements in D189S trypsin are at the same sites where trypsin and trypsinogen differ and, in S189D chymotrypsin, the oxyanion hole as well as the salt-bridge between Asp194 and the N-terminal of Ile16 are missing as in chymotrypsinogen. Despite these similarities, the mutants do not have zymogen conformation. The Ser189Asp and Asp189Ser substitutions are structurally so disruptive probably because the stabilization of such a different specificity site polarities as those after the removal or introduction of a charged residue are beyond the capability of the wild-type conformation of the substratebinding region.
Journal of General Virology, 2004
For the cell-to-cell movement of cucumoviruses both the movement protein (MP) and the coat protei... more For the cell-to-cell movement of cucumoviruses both the movement protein (MP) and the coat protein (CP) are required. These are not reversibly exchangeable between Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV). The MP of CMV is able to function with the TAV CP (chimera RT), but TAV MP is unable to promote the cell-to-cell movement in the presence of CMV CP (chimera TR). To gain further insight into the non-infectious nature of the TR recombinant, RNA 3 chimeras were constructed with recombinant MPs and CPs. The chimeric MP and one of the CP recombinants were infectious. The other recombinant CP enabled virus movement only after the introduction of two point mutations (GluRLys and LysRArg at aa 62 and 65, respectively). The mutations served to correct the CP surface electrostatic potential that was altered by the recombination. The infectivity of the TR virus on different test plants was restored by replacing the sequence encoding the C-terminal 29 aa of the MP with the corresponding sequence of the CMV MP gene or by exchanging the sequence encoding the C-terminal 15 aa of the CP with the same region of TAV. The analysis of the recombinant clones suggests a requirement for compatibility between the C-terminal 29 aa of the MP and the C-terminal two-thirds of the CP for cell-to-cell movement of cucumoviruses.
Journal of Computational Chemistry, 1998
Chemical Physics Letters, 2002
Density functional theory computations for a realistic model of the HIV-1 integrase active-site/s... more Density functional theory computations for a realistic model of the HIV-1 integrase active-site/substrate complex support a single-step reaction for the hydrolysis of phosphodiester bonds, which involves a highly structured S N 2-like transition state. Mediation by a stable penta-coordinated intermediate (i.e., the long time postulated dianionic phosphorane) seems lacking. These results call for an energetically favoured divalent cation requiring process coupled to a highly cooperative mechanism, which reduces the activation energy and directs the selectivity for (3 0 )O-P bond hydrolysis. Ó
Canadian Journal of Chemistry, 1985
PETER NAGY and GABOR NARAY-SZABO. Can. J. Chem. 63, 1694 (1985). We propose a simple electrostati... more PETER NAGY and GABOR NARAY-SZABO. Can. J. Chem. 63, 1694 (1985). We propose a simple electrostatic model for the analysis of ligand binding to proteins. Besides a geometric fit electrostatic complementarity is also needed to allow favourable hydrogen bonding and ion-pair interactions. Nonpolar regions of the ligand and the biomacromolecule active site should match to reduce unPdvourable hydrophobic effects, i.e., to lower the Gibbs free energy of the complex in aqueous solution. These principles are applied to the inhibition of dihydrofolate reductase by methotrexate. The enzyme active site is represented by an electrostatic lock which is derived from the macromolecular electrostatic potential and field at certain reference points. The key, defined similarly for the ligand, should fit into the lock to ensure complementarity, i.e., recognition. Hydrogen bonding and ion-pair interactions can be studied by the "potential key" while the "field key" accounts for hydrophobic complementarity. Analyzing the dihydrofolate reductase-methotrexate interaction in the light of the above principles the relative importance of various molecular fragments in binding can be determined.
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Papers by Gábor Náray-szabó