[Finding an "invisible" binding site for low-molecular compounds on a protein molecule and predicting their inhibiting activity].

Current computational methods have not been able to discover an unknown binding site for low-molecular ligands on a protein receptor and predict parameters of their interaction when this binding site is not distinguished by energy of binding or structural features. Authors propose a method to find an unknown, structurally undefined site for binding low-molecular inhibitors with a protein, as well as to predict kinetic parameters for new compounds using x-ray structure of a protein receptor and experimental interaction constants of a training set of inhibitors. The developed method is applied to discover structural and kinetic parameters of binding C1q, a protein from the first component of complement system, to low-molecular ligands that inhibit its interactions with immune complexes. Authors have suggested that these ligands bind to a region of C1q globular head near residues Arg150 of chain B, and Lys160 and His67 of chain C, supposedly inhibiting the classical pathway of complement activation. Ligands that inhibit interaction of C1q with immune complexes can be used in the therapy of pathological conditions that are related to unwanted complement activation: allergic reactions, xenograft rejection, etc.

[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.

[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.

[A new program complex EFOLD for molecular modeling: the use of a flexible weighting coefficient scheme and the standard valence geometry in modeling the enzyme active sites]. / EFOLD: programmnyi kompleks dlia molekuliarnogo modelirovaniia. Ispol'zovanie nastraivaemoi vesovoi skhemy i standartnoi valentnoi geometrii v modelirovanii aktivnykh tsentrov fermentov.

An algorithm for the representation of biopolymer structures in an internal coordinate system (so-called structure regularization) by minimizing the target function with a flexible weighting coefficient scheme using three components that determine the reliability of deviations of each atom was proposed. For the structure regularization, an algorithm for taking into account the temperature factor was suggested for the first time. It was shown by the example of the aspartyl protease rhizopuspepsin that the representation in the internal coordinate system may result in an accurate reproduction of the structural details of separate molecule fragments, such as the active site region of the enzyme. This algorithm was realized as one of the modules of our EFOLD program complex. The English version of the paper.

[Mechanism of action of aspartic proteinases. V. Conformational characteristics of fragments of substrate-binding sites in rhizopuspepsin and HIV-1 proteinase]. / Mekhanizm deistviia aspartatnykh proteinaz. V. Konformatsionnye vozmozhnosti fragmentov substratsviazyvaiushchikh uchastkov rizopuspepsina i proteinazy HIV-1.

The conformational states of side chains of catalytic Asp residues in active sites of HIV-1 protease and rhizopuspepsin in the potential field of free enzymes were studied by using theoretical conformational analysis. Structural factors that stabilize the conformation of these residues in free enzymes were revealed. Methods of molecular mechanics were used to estimate the stabilization energy of the Met46-Phe53 labile fragments of HIV-1 protease in the potential field of their nearest surrounding amino acid residues for the conformations characteristic of the free protein and similar to that of the protein in enzyme-inhibitor complexes. In solution, the conformational state of the fragments of the free enzyme was concluded to be similar to that observed in the enzyme complex with the ligand and different from that determined by X-ray diffraction analysis. This difference was ascribed to the effect of crystal packing.

[Mechanism of action of aspartic proteases. III. Conformational characteristics of HIV-1 protease inhibitor JG-365]. / Mekhanizm deistviia aspartatnykh proteinaz. III. Konformatsionnye vozmozhnosti ingibitora JG-365 proteinazy HIV-1.

A set of conformations was shown to be characteristic of the free-state spatial structure of substrate-like inhibitor JG-365 for aspartic protease from HIV-1. Among them, the lowest-energy conformations have a folded form of the peptide backbone. The inhibitor has a noncleavable hydroxyethylamine group with an additional chiral center in its structure. Our calculations showed that only the S-isomer of the inhibitor displays conformational characteristics that practically coincide with those of the native substrate for HIV-1 protease. One of the calculated conformations with a completely extended main chain and a relative energy of 9.5 kcal/mol very closely mimics the experimentally observed structure of the inhibitor in the enzyme-inhibitor complex. The realization of this structure is unlikely for a free inhibitor, because it has only a small number of interresidual noncovalent interactions in the extended conformation; these are presumably compensated for by intermolecular interactions at the active site of the enzyme.

[Mechanism of action of aspartic proteases. IV.Conformational characteristics of a substrate and an inhibitor of rhizopuspepsin ]. / Mekhanizm deistviia aspartatnykh proteinaz. IV. Konformatsionnye vozmozhnosti molekul substrata i ingibitora pizopuspepsina.

On the basis of theoretical conformational analysis of separate peptide fragments, the conformational characteristics of two substrates and a substrate-like inhibitor of aspartic protease rhizopuspepsin were studied. It was shown that the spatial structure of these molecules is described by several families of conformations, the transition between which does not require the overcoming of high energy barriers. It was assumed that the stabilization of beta-structural conformations experimentally observed in inhibitor complexes is due to the greater predisposition of extended structures to the formation of effective intermolecular contacts with amino acid residues of the active site of the enzyme.

[Mechanism of action of aspartyl proteinases. VI. Nonvalent enzyme-inhibitory and enzyme-substrate complexes of the aspartyl proteinase rhizopus pepsin]. / Mekhanizm deistviia aspartil'nykh proteinaz. VI. Nevalentnye ferment- ingibitornyi i ferment-substratnyi kompleksy aspartil'noi proteinazy rizopuspepsina.

The structure of a complex of rhizopuspepsin, a fungal aspartyl protease, with Pro1-Phe2-His3-Phe4-psi[CH2-NH]-Phe5-Val6, its substrate-like inhibitor, was calculated by theoretical conformational analysis. The search for energetically favorable conformational variants of the ligand structure was based on the fragmental approach using the dynamic library of peptide fragments, which were successively extended in the potential field of the protein. The root-mean-square deviation of atom positions in the calculated and experimental inhibitor conformations was 0.56 A. A similar approach was used to model a noncovalent complex of rhizopuspepsin with Pro1-Phe2-His3-Lys4-Phe5-Val6, its specific substrate. As a result, two isoenergetic structures of the complex with different arrangements of the cleavable peptide group and a nucleophilic water molecule were calculated. The possibility of the achieving each of these conformations during the catalytic act is considered. It is shown that there are no structural prerequisites for the distortion of the cleavable bond in the active site of the enzyme. On the basis of the resulting structural data, the assumption was made that Asp35 may be protonated at a late stage of formation of the tetrahedral intermediate rather than at the basic state of the complex.

[Mechanism of aspartyl proteinase action. VII. Noncovalent complexes of HIV-1 aspartyl proteinase with substrate and substrate-like inhibitors]. / Mekhanizm deistviia aspartil'nykh proteinaz. VII. Nevalentnye kompleksy aspartil'noi proteinazy HIV-1 s substratom i substratopodobnym ingibitorom.

A computer model of a noncovalent complex of HIV-1 aspartyl protease with substrate-like inhibitor JG-365 was a priori constructed by using the approaches of theoretical conformational analysis and molecular mechanics. The root mean square deviation of the calculated conformation of the inhibitor from the X-ray diffraction analysis data was 0.87 A. These results enabled the a priori calculation of the structure of noncovalent complex of HIV-1 protease with a hexapeptide fragment of its native specific substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val. The only possible orientation of the cleavable peptide bond in this and the nucleophilic water molecule relative to the catalytically active Asp residues of the enzyme (Asp25 and Asp125) was found that provides for the chemical transformation of the substrate to a tetrahedral intermediate. An action mechanism of enzymes of this class was proposed on the basis of the analysis of calculated distances. We showed that neither steric distortion of the cleavable bond nor the formation of unfavorable contacts in molecules of the enzymes and their substrates accompany the optimum orientation of substrate molecules at the active sites of HIV-1 aspartyl proteases and rhizopuspepsin.

[Mechanism of action of aspartic proteinases. 2. Conformational possibilities of an HIV-1 proteinase substrate]. / Mekhanizm deistviia aspartatnykh proteinaz. 2. Konformatsionnye vozmozhnosti molekuly substrata proteinazy HIV-1.

Theoretical conformational analysis of a hexapeptide fragment p17-p24 of the native substrate of the HIV-1 protease was reported. The geometrical and energy parameters of all possible optimal conformations were determined. The data which are necessary for the calculation of the mechanism of the catalytic act of HIV-1 protease were obtained.

[Mechanism of aspartic proteinase action. I. Theory and method]. / Mekhanizm deistviia aspartatnykh proteinaz. !. Teoriia i metod.

Starting from experimental results and contemporary theories of biocatalysis, stereochemistry of aspartic protease functioning was discussed. A general theory of biocatalysis was suggested, which is based on the structural and functional organization of enzyme and substrate molecules and allow a quantitative description of a catalytic act as a continuous, spontaneous, and self-controlled process.