Conserved interactions of the active carboxyls in pepsin-like enzymes and retroviral proteases

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.     

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

On recent evidence, the efficiency of catalysis and the specificity of aspartic proteases depend considerably on the dynamic properties of particular molecular regions and their correlations. Analysis of the three-dimensional structures of these enzymes showed the presence of a continuous chain of hydrogen-bonded groups, which connects regions with highly correlated dynamic parameters and provides for a “cross-hand” interaction between domains. This chain includes the inner oxygens of the active carboxyls and the conserved internal water molecules. The so-called “fireman grip” interdomain hydrogen bonding is part of this chain. Such networks are abortive in retroviral proteases. The role of these interactions in the functions of aspartic proteases is discussed.     

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.     

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.     

Special Features of the Three-Dimensional Structure Defining Properties of Aspartic Proteases

A brief account is given of the specific interactions of some amino acid residues in aspartic proteases of both higher organisms and retroviruses that determine important protease properties: the anomalously low isoelectric point of pepsin and its stability at pH close to 1; the ability of one of the carboxyl groups in the active site of proteases of higher organisms to retain a charged state at any pH value; and the protonated state of another carboxyl, which is necessary for enzymatic activity. It is also explained how such states can be induced in retroviral proteases.     

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.     

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.     

Toward a universal inhibitor of retroviral proteases: comparative analysis of the interactions of LP-130 complexed with proteases from HIV-1, FIV, and EIAV.

One of the major problems encountered in antiviral therapy against AIDS is the emergence of viral variants that exhibit drug resistance. The sequences of proteases (PRs) from related retroviruses sometimes include, at structurally equivalent positions, amino acids identical to those found in drug-resistant forms of HIV-1 PR. The statine-based inhibitor LP-130 was found to be a universal, nanomolar-range inhibitor against all tested retroviral PRs. We solved the crystal structures of LP-130 in complex with retroviral PRs from HIV-1, feline immunodeficiency virus, and equine infectious anemia virus and compared the structures to determine the differences in the interactions between the inhibitor and the active-site residues of the enzymes. This comparison shows an extraordinary similarity in the binding modes of the inhibitor molecules. The only exceptions are the different conformations of naphthylalanine side chains at the P3/P3' positions, which might be responsible for the variation in the Ki values. These findings indicate that successful inhibition of different retroviral PRs by LP-130 is achieved because this compound can be accommodated without serious conformational differences, despite the variations in the type of residues forming the active-site region. Although strong, specific interactions between the ligand and the enzyme might improve the potency of the inhibitor, the absence of such interactions seems to favor the universality of the compound. Hence, the ability of potential anti-AIDS drugs to inhibit multiple retroviral PRs might indicate their likelihood of not eliciting drug resistance. These studies may also contribute to the development of a small-animal model for preclinical testing of antiviral compounds.     

Systematics in the interaction of metal ions with the main-chain carbonyl group in protein structures

An analysis of the geometry of metal binding by peptide carbonyl groups in proteins is presented. Such metal ions are predominantly calcium in known protein structures. Cations tend to be located in the peptide plane, near the C = O bond direction. This distribution differs from that observed for water molecules bound to carbonyl oxygens. Most metal ions are bound to carbonyl oxygens of peptides in turns or in regions with no regular secondary structure. More infrequent binding interactions occur at the C-terminal end of alpha-helices or at the edges and sides of beta-sheets, where the geometrical preferences of the metal-carbonyl interaction may be satisfied. In many proteins carbonyl groups that are one, two, or three residues apart along the polypeptide chain bind to the same cation; these structures show a limited number of main-chain conformations around the metal center.     

Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease

The pepsin-like aspartyl proteases consist of a single polypeptide chain with topologically similar amino- and carboxyl-terminal domains, each of which contributes 1 aspartic acid residue to the active site. This structure has been proposed to have evolved by gene duplication and fusion from a dimeric enzyme composed of two identical polypeptide chains, such as the aspartyl protease (PRT) of human immunodeficiency virus type 1 (HIV-1). To determine if a single polypeptide form of the HIV-1 protease would be enzymatically active, two protease coding regions were linked to form a dimeric gene (pFGGP). Expression of this gene in Escherichia coli yielded a protein with the expected molecular mass of 22 kDa. The in vitro kinetic parameters of PRT and FGGP (where FGGP is the single polypeptide form of the HIV-1 protease with 2 glycine residues connecting the two subunits) for three peptide substrates are similar. Construction and analysis of a CheY-GAG-FGGP fusion protein demonstrated that FGGP is capable of precursor processing in vivo. Mutation of one or both of the active site aspartates to either asparagine or glutamate rendered the enzyme inactive, demonstrating that both active site aspartate residues are required for enzymatic activity.     

Characterization of two hydrophobic methyl clusters in HIV-1 protease by NMR spin relaxation in solution

Nearly 50 % of the amino acid residues of HIV-1 protease contain methyl side-chains, most of which appear to be organized into two clusters: the inner cluster that nearly surrounds the active site and the outer cluster that contains the hydrophobic core which stabilizes the inhibitor-free protease structure. NMR relaxation experiments sensitive to motions of methyl groups on the sub-nanosecond and the milli-microsecond time-scales revealed flexible methyl groups in residues that link the two clusters, the methyl groups of L10, L23, V75, and L76. We hypothesize that flexibility at the junctions of these clusters allows the protease to minimize conformational changes upon drug-binding. The two-methyl cluster motif appears to be a common structural feature among retroviral proteases and may play a similar role throughout this family of enzymes.     

A novel modified peptide derived from membrane‐proximal external region of human immunodeficiency virus type 1 envelope significantly enhances retrovirus infection

Efficient gene transfer is a critical goal in retroviral transduction. Several peptides capable of forming amyloid fibrils, such as the 39‐residue semen‐derived infection‐enhancing peptide (SEVI), have demonstrated the ability to boost retroviral gene delivery. Here, a 13‐residue peptide P13 (Ac‐⁶⁷¹NWFDITNWLWYIK⁶⁸³) derived from the membrane‐proximal external region of the human immunodeficiency virus type 1 (HIV‐1) gp41 transmembrane protein, together with its 16‐residue peptide derivative (P16) were found to enhance HIV‐1 infection significantly. Both peptides, P13 and P16, could form amyloid fibril structures to potently enhance HIV‐1 infectivity. Further investigations showed that both aromatic Trp residues and cationic Lys residues contributed to the enhancement of HIV‐1 infection by these two active peptides. P16 could more effectively augment HIV‐1 YU‐2 infection than SEVI, implying its potential applications as a tool in the lab to improve gene transfer rates. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Structural analysis of Pla protein from the biological warfare agent Yersinia pestis: docking and molecular dynamics of interactions with the mammalian plasminogen system

Yersinia pestis protein Pla is a plasmid-coded outer membrane protein with aspartic-protease activity. Pla exhibits a plasminogen (Plg) activator activity (PAA) that promotes the cleavage of Plg to the active serine-protease form called plasmin. Exactly how Pla activates Plg into plasmin remains unclear. To investigate this event, we performed the interactions between the predicted Plg and Pla protein structures by rigid-body docking with the HEX program and evaluated the complex stability by molecular dynamics (MD) using the GROMACS package programs. The predicted docked complex of Plg–Pla shows the same interaction site predicted by experimental site-direct mutagenesis in other studies. After a total of 8?ns of MD simulation, we observed the relaxation of the beta-barrel structure of Pla and the progressive approximation and stabilization between the cleavage site of Plg into the extracellular loops of Pla, followed by the increase in the number of H bonds. We also report here the aminoacids that participate in the active site and the sub sites of interaction. The total understanding of these interactions can be an important tool for drug design against bacterial proteases.     

Tobacco Etch Virus Protease: Crystal Structure of the Active Enzyme and Its Inactive Mutant

Tobacco Etch Virus Protease (TEV protease) is widely used as a tool for separation of recombinant target proteins from their fusion partners. The crystal structures of two mutants of TEV protease, the active autolysis-resistant mutant TEV-S219D in complex with the proteolysis product, and the inactive mutant TEV-C151A in complex with a substrate, have been determined at 1.8 and 2.2 Å resolution, respectively. The active sites of both mutants, including their oxyanion holes, have identical structures. The C-terminal residues 217–221 of the enzyme are involved in formation of the binding pockets S ₃–S ₆. This indicates that the autolysis of the peptide bond Met218–Ser219 exerts a strong effect on the fine-tuning of the substrate in the enzyme active site, which results in a considerable decrease in the enzymatic activity.     

Bioinformatics and molecular modeling in chemical enzymology. Active sites of hydrolases

Comparison and multiple alignments of amino acid sequences of a representative number of related enzymes demonstrate the existence of certain positions of amino acid residues which are permanently reproducible in all members of the whole family. The use of the bioinformatic approach revealed conservative residues in each of the related enzymes and ranked amino acid conservatism for the overall enzymatic catalysis. Glycine and aspartic acid residues were shown to be the most essential for structure and catalytic activity of enzymes. Amino acid residues forming catalytic subsite of the active site of enzymes are always highly conservative. Analysis revealed that aspartic acid carboxyl group is the most frequently employed nucleophilic (in deprotonated form) and electrophilic (in protonated form) agent involved in activation of molecules by the mechanism of general base and acidic catalyses in the catalytic sites of enzymes. Glycine is a unique amino acid possessing the highest possibilities for rotation along C–C and C–N bonds of the polypeptide chain. The conservative fixation of the glycine residue in polypeptide chains of related enzymes provides a possibility for directed assembly of amino acid residues into the catalytic subsite structure. It is possible that the conservative glycines provide known conformational mobility of the protein and the active site. Methods of molecular modeling were used for analysis of structural substitutions of conservative and non-conservative glycines and their effects on geometry of catalytic site of typical hydrolases. The substitution of glycine(s) for alanine significantly altered the catalytic site structures.     

Closing of the flaps of HIV-1 protease induced by substrate binding: a model of a flap closing mechanism in retroviral aspartic proteases

The active site of aspartic proteases is covered by one or more flaps, which control access to the active site and play a significant role in the binding of the substrate. An extensive conformational change of the flaps takes place upon binding of substrate to the active site. A long molecular dynamics simulation was performed on the complex consisting of a peptide (CA-p2) from a natural substrate cleavage site of the gag/pol polyprotein placed in the active site of HIV-1 protease (PR) with an open flap conformation. During the simulation, the substrate induced the closing of the flaps into the closed conformation in an asymmetrical way through a hydrophobic intermediate state cluster. The nature of the residues of HIV-1 PR identified to be important in the flap closing mechanism is conserved across known structures of retroviral aspartic proteases family. The flap closing mechanism described in HIV-1 PR is proposed to be a general model for flap closing in retroviral aspartic proteases.     

Comparison of inhibitor binding in HIV-1 protease and in non-viral aspartic proteases: the role of the flap

The crystal structure of HIV-1 protease with an inhibitor has been compared with the structures of non-viral aspartic proteases complexed with inhibitors. In the dimeric HIV-1 protease, two 4-stranded beta-sheets are formed by half of the inhibitor, residues 27-29, and the flap from each monomer. In the monomeric non-viral enzyme the single flap does not form a beta-sheet with an inhibitor. The HIV-1 protease shows more interactions with a longer peptide inhibitor than are observed in non-viral aspartic protease-inhibitor complexes. This, and the large movement of the flaps, restricts the conformation of the protease cleavage sites in the retroviral polyprotein precursor.     

How the features of three-dimensional structure of aspartate proteinases determine their properties

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.     

Crystal structures of human mitochondrial branched chain aminotransferase reaction intermediates: ketimine and pyridoxamine phosphate forms

The three-dimensional structures of the isoleucine ketimine and the pyridoxamine phosphate forms of human mitochondrial branched chain aminotransferase (hBCATm) have been determined crystallographically at 1.9 A resolution. The hBCATm-catalyzed transamination can be described in molecular terms together with the earlier solved pyridoxal phosphate forms of the enzyme. The active site lysine, Lys202, undergoes large conformational changes, and the pyridine ring of the cofactor tilts by about 18 degrees during catalysis. A major determinant of the enzyme's substrate and stereospecificity for L-branched chain amino acids is a group of hydrophobic residues that form three hydrophobic surfaces and lock the side chain in place. Short-chain aliphatic amino acid side chains are unable to interact through van der Waals contacts with any of the surfaces whereas bulky aromatic side chains would result in significant steric hindrance. As shown by modeling, and in agreement with previous biochemical data, glutamate but not aspartate can form hydrogen bond interactions. The carboxylate group of the bound isoleucine is on the same side as the phosphate group of the cofactor. These active site interactions are largely retained in a model of the human cytosolic branched chain aminotransferase (hBCATc), suggesting that residues in the second tier of interactions are likely to determine the specificity of hBCATc for the drug gabapentin. Finally, the structures reveal a unique role for cysteine residues in the mammalian BCAT. Cys315 and Cys318, which immediately follow a beta-turn (residues 311-314) and are located just outside the active site, form an unusual thiol-thiolate hydrogen bond. This beta-turn positions Thr313 for its interaction with the pyridoxal phosphate oxygens and substrate alpha-carboxylate group.     

Shewasin A, an active pepsin homolog from the bacterium Shewanella amazonensis

The view has been widely held that pepsin-like aspartic proteinases are found only in eukaryotes, and not in bacteria. However, a recent bioinformatics search [Rawlings ND & Bateman A (2009) BMC Genomics10, 437] revealed that, in seven of ～ 1000 completely sequenced bacterial genomes, genes were present encoding polypeptides that displayed the requisite hallmark sequence motifs of pepsin-like aspartic proteinases. The implications of this theoretical observation prompted us to generate biochemical data to validate this finding experimentally. The aspartic proteinase gene from one of the seven identified bacterial species, Shewanella amazonensis, was expressed in Escherichia coli. The recombinant protein, termed shewasin A, was produced in soluble form, purified to homogeneity, and shown to display properties remarkably similar to those of pepsin-like aspartic proteinases. Shewasin A was maximally active at acidic pH values, cleaving a substrate that has been widely used for assessment of the proteolytic activity of other aspartic proteinases, and displayed a clear preference for cleaving peptide bonds between hydrophobic residues in the P1*P1' positions of the substrate. It was completely inhibited by the general inhibitor of aspartic proteinases, pepstatin, and mutation of one of the catalytic Asp residues (in the Asp-Thr-Gly motif of the N-terminal domain) resulted in complete loss of enzymatic activity. It can thus be concluded unequivocally that this Shewanella gene encodes an active pepsin-like aspartic proteinase. It is now beyond doubt that pepsin-like aspartic proteinases are not confined to eukaryotes, but are encoded within some species of bacteria. The distinctions between the bacterial and eukaryotic polypeptides are discussed and their evolutionary relationships are outlined.