[Modeling of substrate and inhibitory complexes of histidine-aspartic protease].

A three-dimensional structure of histo-aspartic protease (HAP), a pepsin-like enzyme from the causative agent of malaria Plasmodium falciparum, is suggested on the basis of homologous modeling followed by equilibration by the method of molecular dynamics. The presence of a His residue in the catalytic site instead of an Asp residue, which is characteristic of pepsin-like enzymes, and replacement of some other conserved residues in the active site make it possible for the enzyme to function by the covalent mechanism inherent in serine proteases. The detailed structures of HAP complexes with pepstatin, a noncovalent inhibitor of aspartic proteases, and phenylmethylsulfonyl fluoride, a covalent inhibitor of serine proteases, as well as with a pentapeptide substrate are discussed.

[Interdomain interactions in aspartic proteases of higher organisms and their analogs in retroviral enzymes].

A continuous chain of hydrogen bonded groups, which forms cross-hands interaction between domains in molecules of pepsin-like enzymes, has been revealed. The chain contains a pair of 6 symmetrically related hydrogen bonds between main chain atoms and the two conserved water molecules. The peptide groups forming hydrogen bond with the inner oxygens of the active carboxyls are important elements of the chain. The so-called "fireman grip" hydrogen bonding, consisting of a pair of the two symmetrically related bonds, is an integral part of this system of interactions. One of the water molecules in this system has a zero accessibility and forms a very short hydrogen bond with the active site interacting peptide group. This chain connects tightly the two regions of domains which have a high correlation in conformational mobility. The retroviral enzymes have an abortive chain of the interdomain interaction in this region which is reduced to the "fireman grip" net.

Molecular and crystal structures of monoclinic porcine pepsin refined at 1.8 A resolution.

The molecular structure of the archetypal aspartic proteinase, porcine pepsin (EC 3.4.23.1), has been refined using data collected from a single monoclinic crystal on a twin multiwire detector system to 1.8 A resolution. The current crystallographic R-factor (= sigma parallel to Fo/-/Fc parallel to/sigma/Fo/) is 0.174 for the 20,519 reflections with /Fo/ greater than or equal to 3 sigma (Fo) in the range 8.0 to 1.8 A (/Fo/ and /Fc/ are the observed and calculated structure factor amplitudes respectively). The refinement has shown conclusively that there are only 326 amino acid residues in porcine pepsin. Ile230 is not present in the molecule. The two catalytic residues Asp32 and Asp215 have dispositions in porcine pepsin very similar to the dispositions of the equivalent residues in the other aspartic proteinases of known structure. A bound solvent molecule is associated with both carboxyl groups at the active site. No bound ethanol molecule could be identified conclusively in the structure. The average thermal motion parameter of the residues that comprise the C-terminal domain of pepsin is approximately twice that of the residues in the N-terminal domain. Comparisons of the tertiary structure of pepsin with porcine pepsinogen, penicillopepsin, rhizopus pepsin and endothia pepsin reveal that the N-terminal domains are topographically more similar than the conformationally flexible C-terminal domains. The conformational differences may be modeled as rigid-body movements of "reduced" C-terminal domains (residues 193 to 212 and 223 to 298 in pepsin numbering). A similar movement of the C-terminal domain of endothia pepsin has been observed upon inhibitor binding. A phosphoryl group covalently attached to Ser68 O gamma has been identified in the electron density map of porcine pepsin. The low pKa1 value for this group, coupled with unusual microenvironments for several of the aspartyl carboxylate groups, ensures a net negative charge on porcine pepsin in a strongly acid medium. Thus, there is a structural explanation for the very early observations of "anodic migration" of porcine pepsin at pH 1. In the crystals, the molecules are packed tightly into a monoclinic unit cell. There are 190 direct contacts (less than or equal to 4.0 A) between a central pepsin molecule and the five unique symmetry-related molecules surrounding it in the crystalline lattice. The tight packing in this cell makes pepsin's active site and binding cleft relatively inaccessible to substrate analogs or inhibitors.

Analysis of crystal structures of aspartic proteinases: on the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes.

To elucidate the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes, we analyzed and compared the crystal structures of these enzymes, their complexes with inhibitors, and zymogens in the active site area (a total of 82 structures). In addition to the water molecule (W1) located between the active carboxyls and playing a role of the nucleophile during catalytic reaction, another water molecule (W2) at the vicinity of the active groups was found to be completely conserved. This water molecule plays an essential role in formation of a chain of hydrogen-bonded residues between the active site flap and the active carboxyls on ligand binding. These data suggest a new approach to understanding the role of residues around the catalytic site, which can assist the development of the catalytic reaction. The influence of groups adjacent to the active carboxyls is manifested by pepsin activity at pH 1.0. Some features of pepsin-like enzymes and their mutants are discussed in the framework of the approach.

Possible role of some groups in the structure and function of HIV-1 protease as revealed by molecular modeling studies.

Retroviral proteases belong to the class of aspartic proteases. A molecular model of HIV-1 protease has been built on the basis of the consensus template specific for the domains of these enzymes. The template region comprises more than a half of the HIV-1 protease monomer structure, it includes the active site, formed at the junction of the two monomers, binding pockets of the enzyme, and some other molecular segments. These regions can be more conveniently described than other parts of the structure. Some properties of the HIV-1 protease molecule are discussed, as well as of probable inhibitors. The properties of the model structure are in good agreement with the recent results of crystallographic studies of Rous sarcoma virus protease.

Structure of glutamate decarboxylase and related PLP-enzymes: computer-graphical studies.

Amino acid sequences of E. coli glutamate decarboxylase (GADa) and those of 36 GAD of different origin were compared by pairwise alignment using computer program CLUSTAL. GADalpha and plant enzymes showed 59.8-67.8% subunit homology, GADalpha and other bacterial GAD--49.8-77.6%, whereas GADalpha and animal enzymes--13.9-58.8%. Two PLP domains exhibited higher homology comparing to that of the whole subunit in the case of GAD67, plant (68.4-73.9%), and bacterial (46.7-83.2%) enzymes. The alignment of PLP-domains of 37 GAD, three group II decarboxylases, and two pyridoxal enzymes with known 3D structures (bacterial ORD and mAAT from chicken heart) allowed us to reveal conserved residues of the active sites. Their functional role is discussed. Modelling of the PLP-binding sites in active centers for GADalpha and human brain GAD67 was done using the Swiss-PdbViewer homology modelling program. Although the homology between GADalpha and GAD67 is rather low, structural similarity of their active sites allows us to consider here a functional convergence. Thus, glutamate decarboxylation by GADalpha may be helpful for understanding general mechanism of this reaction.

Some aspects of structural studies on aspartic proteinases.

This paper gives a brief overview over the differences and similarities in the structure of aspartic proteinases presently available. Comparison of the three-dimentional structure of different aspartic proteinases by a common intramolecular coordinate system have been performed. The intramolecular movable subdomains have been localized and the role of motion in substrate binding and zymogen activation is discussed.

[How and why is pepsin stable and active at pH 2?]. / Zachem i pochemu pepsin stabilen i aktiven pri pH 2?

The HCl in the mammalian stomach is concentrated enough to digest the stomach itself and to cause denaturation of proteins. The paper summarize studies which explain why the gastric epithelium remains undamaged and gastric proteinase pepsin has the most stable and active structure at such extreme conditions. Pepsin is the first proteinase which starts protein proteolysis during the multistep process of protein digestion, and it splits mainly their hydrophobic cores unfolded in acidic media. Data on the disposition of the charged groups in the three-dimension structure of pepsin, which explain the extraordinary properties of the enzyme, are discussed.

[The structure of pepsin. I. Molecular self-symmetry of the enzyme and implications for the evolution of aspartate proteinases]. / Struktura pepsina. I. Sobstvennaia simmetriia molekuly fermenta i vozmozhnye puti évoliutsii aspartatnykh proteinaz.

This is the first paper in the series of publications on the detailed description of different aspects of pepsin structure. It concerns the domain structure of the enzyme and the topology of the arrangement of beta-strands in pepsin and related aspartic proteases, this topology being quite different from that of all other beta-proteins. The topology, molecular symmetry and the close proximity of the N- and C-termini of domains suggest that at the first stages of evolution polypeptide chains of some preproteins could be closed in rings, which become open at the subsequent stages. Their genes duplicated twice, and the fusion process resulted in a gene consisting of similar parts.

[Conserved interactions of the active carboxyls in pepsin-like enzymes and retroviral proteases]. / Konservativnye vzaimodeistviia aktivnykh karboksilov v pepsinopodobnykh i retrovirusnykh proteazakh.

In addition to previous studies, 30 crystal structures of retroviral proteases corresponding to the highest resolution were inspected to analyze the interactions of the active carboxyl with surroundings groups. The outer oxygen of the active carboxyl in retroviral enzymes form contacts only with the water molecule participating in catalysis. This is an important difference between retroviral proteases and pepsin-like enzymes, which form a net of hydrogen bonds of these outer oxygen with residues neighboring the catalytic site in 3D structures. At the same time, it was found that in all aspartic proteases the inner oxygen of the active carboxyl are also involved in the chain of interactions through peptide groups Thr-Gly adjacent to the active residues. Polarization of these peptide groups influences the donor-acceptor properties of the active carboxyl. The found chain of interactions is more extensive in retroviral than in pepsin-like proteases; however, its main part is conserved for the whole class of these enzymes. Some implications of the role of these interactions are discussed.

[The structure of pepsin. II. Structure of the enzyme active site (at 2 angstroms resolution)]. / Struktura pepsina. II. Stroenie aktivnogo tsentra fermenta (razreshenie 2 angstrom).

Detailed structure of the pepsin active site in the region of the active aspartic acid residues and substrate binding S1 and S1' sites is considered. At the active site of the enzyme crystals studied several molecules of ethanol were detected, which interact with active groups. The catalytic properties of aspartyl proteinases towards dipeptide substrates were explained on the base of the specific structure of S1 and S1' binding sites.

[The role of peripheral interactions in chymosin specificity]. / O roli perifericheskikh vzaimodeistvii v spetsifichnosti khimozina.

Kinetic parameters for the splitting of model peptide substrates and chi-casein with chymosin have been interpreted on the basis of the three-dimensional structure of chymosin. Model peptide substrates contain a fragment of the chi-casein sequence in the region of the bond Phe-105--Met-106 splitted with the enzyme. It was shown that the possible reason of the enormous milk-clotting efficiency of chymosin may be partly associated with the electrostatic interaction of the positive charged segment 98-102 (His-Pro-His-Pro-His) of the substrate and outer loop of the enzyme which contains Glu-245, Asp-247, Asp-249, Asp-251.

[Molecular dynamics analysis of chymosin conformations in solution and in crystalline environment]. / Molekuliarno-dinamicheskii analiz konformatsii khimozina v rastvore i v kristalle.

The method of molecular dynamics in explicit solvent was applied to test the hypothesis of the existence of a self-inhibited form of chymosin in solution. The paths and energies were calculated for chymosin in solution and in a crystalline environment. The modeling revealed that the intermolecular contacts of chymosin in crystal have negligible influence on the energy stabilization of its self-inhibited conformation. On the other hand, upon molecular dynamics simulation of the active and self-inhibited forms in solution their conformational energies proved to be quite close and the potential barrier between them relatively low. All this supports the possibility of chymosin to adopt spontaneously the self-inhibited conformation in solution, and indicates that it is one of the really existing enzyme forms rather than a crystal packing artifact. The results obtained open novel approaches to studying the specificity of chymosin as well as other aspartic proteinases.

[X-ray study of chymosin. I. Molecular replacement at a 3 angstroms resolution]. / Rentgenostrukturnye issledovaniia khimozina. I. Molekuliarnoe zameshchenie pri razreshenii 3 angstrom.

The determination of the three dimensional structure of chymosin at 3 A resolution by molecular replacement method is described. The rotation functions for various aspartic proteases were calculated and combined results were used for the refinement of orientational parameters of chymosin molecules in the unit cell. The interpretation of Crowther-Blow translation function map with packing consideration enable to place correctly the molecules in the chymosin unit cell. Several difference Fourier syntheses for chymosin were calculated and differences between pepsin and chymosin structures were detected.

[Theoretical studies of the electrostatic interactions in aspartic proteinases, intramolecular interactions in pepsin and penicillopepsin]. / Teoreticheskie issledovaniia élektrostaticheskikh vzaimodeistvii v aspartatnykh proteinazakh, vnutrimolekuliarnye vzaimodeistviia v pepsine i penitsillopepsine.

A semi-empirical approach has been used to estimate the intramolecular electrostatic interactions in pepsin and penicillopepsin. The pH-dependence of the free energy electrostatic term was calculated, and the pH-dependence of the domain interactions has been estimated. As it was shown, the contribution of electrostatic interactions is rather small for the stabilization of the native structure. At the same time the electrostatic repulsion between domains increases with the increase of pH. The later can be the cause of the alkaline denaturation of pepsin and domain mobility.

[X-ray analysis of pepsin. VI. Atomic structure of the enzyme at 2-angstrom resolution]. / Rentgenostrukturnyi analiz pepsina. VI. Atomnaia struktura fermenta pri razreshenii 2 angstrom.

The crystallographic refinement of pepsin structure at 2 A resolution is described. Real space refinement and Jack and Levitt methods were used. As a result, the refined atomic coordinates of 2436 nonhydrogen atoms were obtained. Values of crystallographic R-factor and conformational energy are 29.2% and -1347 kcal/mol correspondingly. The most important and interesting features of pepsin structure are discussed.

[X-ray structural analysis of pepsin. V. Conformation of the main chain of the enzyme]. / Rentgenostrukturnyi analiz pepsina. V. Konformatsiia glavnoi tsepi fermenta.

A detailed description of structural investigations of pepsin from 3.5 to 2.7 A resolution are given. The main attention is drawn to the conformation of the polypeptide backbone of the enzyme. The numbers of amino acid residues involved in the formation of various structural elements are listed. The structure of pepsin is similar to that of acid proteases isolated from lower organisms. The two domain structure of all acid proteases has a periodicity in the sequence of beta-segments and helices and a very specific symmetrical structure of each domain. This makes it possible to describe the structure of acid proteases in simple terms.

[Various aspects of structural studies of aspartate proteinases]. / Nekotorye aspekty strukturnykh issledovanii aspartatnykh proteinaz.

The paper is a brief account of aspartic proteinases' structural studies developed in V.A. Engelhardt Institute of Molecular Biology during the last 3 years. The work on porcine pepsin has been finalized after the refinement of the monoclinic crystal form at 1.8 A resolution performed in collaboration with the group of protein structure and function studies of the University of Alberta in Canada. An important structural property of chymosin which explains the enzyme specificity has been found. Protein engineering work on chymosin is being developed. The structural template for aspartic proteinases has been elucidated and on the basis of this template the model of HIV-1 protease molecule has been built. Some approaches to the design of HIV-1 protease inhibitors were elucidated.

[How the features of three-dimensional structure of aspartate proteinases determine their properties]. / Kak osobennosti trekhmernoi struktury aspartatnykh proteinaz opredeliaiut ikh svoistva.

There is given a brief account of the specific interactions of some amino acid residues in aspartic proteases of both higher organisms and retroviruses that determine their important properties: an anomalously low isoelectric point of pepsin and its stability at pH close to unity; the ability of one of the carboxyl groups in the active site of proteases of higher organisms to retain charged state at any pH value and protonated state of another carboxyl, which is necessary for their enzymatic activity. It is also explained how such states can be induced in retroviral proteases.