Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.     

X-ray structure of HIV-1 protease in situ product complex

HIV-1 protease is an effective target for design of different types of drugs against AIDS. HIV-1 protease is also one of the few enzymes that can cleave substrates containing both proline and nonproline residues at the cleavage site. We report here the first structure of HIV-1 protease complexed with the product peptides SQNY and PIV derived by in situ cleavage of the oligopeptide substrate SQNYPIV, within the crystals. In the structure, refined against 2.0-A resolution synchrotron data, a carboxyl oxygen of SQNY is hydrogen-bonded with the N-terminal nitrogen atom of PIV. At the same time, this proline nitrogen atom does not form any hydrogen bond with catalytic aspartates. These two observations suggest that the protonation of scissile nitrogen, during peptide bond cleavage, is by a gem-hydroxyl of the tetrahedral intermediate rather than by a catalytic aspartic acid.     

Enzymatic and chemical properties of an endopeptidase from the larva of the hornet Vespa crabro.

An endopeptidase from the larvae of the hornet Vespa crabro has been purified to homogeneity. The enzyme has been characterized with respect to molecular weight, amino acid compositon, and amino- and carboxyl-terminal sequences. The catalytic properties of the hornet protease are similar to those of bovine chymotrypsin with respect to inactivation by phenylmethanesulfonyl fluoride and carbobenzoxyphenylalanine chloro ketone and preferential peptide bond cleavage at aromatic amino acid residues. In contrast to bovine chymotrypsin, the hornet protease is not inhibited by the basic pancreatic Kunitz inhibitor, soybean inhibitor, or chicken ovomucoid. The molecular weight, as determined by several independent methods, was found to be 14 500. The protease is a single-chain protein containing two disulfide bonds. The terminal sequences are: NH2-Ile-Val-Gly-Gly-Ile-Asp.....Gly-Lys-Tyr-Pro-Tyr-Gln-Val-Ser-Leu-Arg-COOH.     

Cleavage specificity of the serine protease of Aeromonas sobria, a member of the kexin family of subtilases

Subtilisin-like proteases have been grouped into six families based on a sequence of the catalytic domain. One of the six is the kexin family, of which furin is a representative protease. All members of the kexin family, except one, are from eukaryotes. The one prokaryotic protease is a serine protease of Aeromonas sorbria (ASP). Here, we examined the substrate specificity of ASP based on the cleavage of short peptides. The results showed that ASP preferentially cleaves the peptide bond following two basic residues, one of which is Lys, but not the bond following a single basic residue. This indicates that the tertiary structure around the catalytic domain of ASP resembles, but is not identical to that of furin. Prekallikrein was cleaved into four fragments by ASP, indicating that the protein must be cleaved at specific sequences.     

Molecular mechanics calculations on Rous sarcoma virus protease with peptide substrates.

Molecular models of Rous sarcoma virus (RSV) protease and 20 peptide substrates with single amino acid substitutions at positions from P4 to P3', where the scissile bond is between P1 and P1'. were built and compared with kinetic measurements. The unsubstituted peptide substrate. Pro-Ala-Val-Ser-Leu-Ala-Met-Thr, represents the NC-PR cleavage site of RSV protease. Models were built of two intermediates in the catalytic reaction, RSV protease with peptide substrate and with the tetrahedral intermediate. The energy minimization used an algorithm that increased the speed and eliminated a cutoff for nonbonded interactions. The calculated protease-substrate interaction energies showed correlation with the relative catalytic efficiency of peptide hydrolysis. The calculated interaction energies for the 8 RSV protease-substrate models with changes in P1 to P1' next to the scissile bond gave the highest correlation coefficient of 0.79 with the kinetic measurements, whereas all 20 substrates showed the lower, but still significant correlation of 0.46. Models of the tetrahedral reaction intermediates gave a correlation of 0.72 for the 8 substrates with changes next to the scissile bond, whereas a correlation coefficient of only 0.34 was observed for all 20 substrates. The differences between the energies calculated for the tetrahedral intermediate and the bound peptide gave the most significant correlation coefficients of 0.90 for models with changes in P1 and P1', and 0.56 for all substrates. These results are compared to those from similar calculations on HIV-1 protease and discussed in relation to the rate-limiting steps in the catalytic mechanism and the entropic contributions.     

The hydrolysis and resynthesis of a single reactive site peptide bond in recombinant antistasin by coagulation factor Xa.

Antistasin (ATS) is a 119-amino acid, leech-derived protein which exhibits selective, tight-binding inhibition of blood coagulation factor Xa. Prolonged incubation of ATS with factor Xa leads to the highly specific hydrolysis of the peptide bond between residues Arg34 and Val35, implicating this peptide bond as the putative reactive site. We report here the preparation of pure, cleaved (modified) recombinant ATS (rATS) and utilize this material to provide additional proof that the cleaved peptide bond is in fact the reactive site. Modified rATS retains strong inhibitory potency against factor Xa as evidenced by a dissociation constant of 166.3 +/- 9.6 pM; four-fold greater than that of native inhibitor, 43.4 +/- 1.4 pM. Incubation of pure, modified rATS with catalytic amounts of factor Xa results in resynthesis of the hydrolyzed peptide bond, achieving an equilibrium near unity between native and modified inhibitors. Specific removal of the newly formed carboxy-terminal Arg residue from modified rATS by carboxypeptidase B treatment obviates its conversion to native inhibitor coincident with the complete loss of inhibitory activity. These results establish that rATS inhibits factor Xa according to a standard mechanism of serine protease inhibitors and support the contention that the Arg34-Val35 peptide bond constitutes the reactive site.     

Reaction of LexA repressor with diisopropyl fluorophosphate. A test of the serine protease model

The LexA repressor of Escherichia coli modulates the expression of the SOS regulon. In the presence of DNA damaging agents in vivo, the 202-amino acid LexA repressor is inactivated by specific RecA-mediated cleavage of the Ala-84/Gly-85 peptide bond. In vitro. LexA cleavage requires activated RecA at neutral pH, and proceeds spontaneously at high pH in an intramolecular reaction termed autodigestion. A model has been proposed for the mechanism of autodigestion in which serine 119 serves as the reactive nucleophile that attacks the Ala-84/Gly-85 peptide bond in a manner analogous to a serine protease, while uncharged lysine 156 activates the serine 119 hydroxyl group. In this work, we have tested this model by examining the effect of the serine protease inhibitor diisopropyl fluorophosphate (DFP) on autodigestion. We found that DFP inhibited autodigestion and that serine 119 was the only serine residue to react with DFP. We also examined [3H]DFP incorporation by a number of cleavage-impaired LexA mutant proteins and found that mutations in the proposed active site, but not in the cleavage site, significantly reduced the rate of [3H]DFP incorporation. Finally, we showed that the purified carboxyl-terminal domain, which contains the proposed catalytic residues, incorporated [3H]DFP at a rate indistinguishable from the intact protein. These data further support our current model for the mechanism of autodigestion and the organization of LexA.     

Catalytic Water Co-Existing with a Product Peptide in the Active Site of HIV-1 Protease Revealed by X-Ray Structure Analysis

Background

It is known that HIV-1 protease is an important target for design of antiviral compounds in the treatment of Acquired Immuno Deficiency Syndrome (AIDS). In this context, understanding the catalytic mechanism of the enzyme is of crucial importance as transition state structure directs inhibitor design. Most mechanistic proposals invoke nucleophilic attack on the scissile peptide bond by a water molecule. But such a water molecule coexisting with any ligand in the active site has not been found so far in the crystal structures.

Principal Findings

We report here the first observation of the coexistence in the active site, of a water molecule WAT1, along with the carboxyl terminal product (Q product) peptide. The product peptide has been generated in situ through cleavage of the full-length substrate. The N-terminal product (P product) has diffused out and is replaced by a set of water molecules while the Q product is still held in the active site through hydrogen bonds. The position of WAT1, which hydrogen bonds to both the catalytic aspartates, is different from when there is no substrate bound in the active site. We propose WAT1 to be the position from where catalytic water attacks the scissile peptide bond. Comparison of structures of HIV-1 protease complexed with the same oligopeptide substrate, but at pH 2.0 and at pH 7.0 shows interesting changes in the conformation and hydrogen bonding interactions from the catalytic aspartates.

Conclusions/Significance

The structure is suggestive of the repositioning, during substrate binding, of the catalytic water for activation and subsequent nucleophilic attack. The structure could be a snap shot of the enzyme active site primed for the next round of catalysis. This structure further suggests that to achieve the goal of designing inhibitors mimicking the transition-state, the hydrogen-bonding pattern between WAT1 and the enzyme should be replicated.     

Soluble oligomers of the intramembrane serine protease YqgP are catalytically active in the absence of detergents

Rhomboid, a polytopic membrane serine protease, represents a unique class of proteases that cleave substrates within the transmembrane domain. Elucidating the mechanism of this extraordinary catalysis comes with inherent challenges related to membrane-associated peptide hydrolysis. Here we established a system that allows expression and isolation of YqgP, a rhomboid homologue from Bacillus subtilis, as a soluble protein. Intriguingly, soluble YqgP is able to specifically cleave a peptide substrate that contains the transmembrane domain of Spitz. Mutation of the catalytic dyad abolished protease activity, and substitution of another highly conserved residue, Asn241, with Ala or Asp significantly reduced the catalytic efficiency of YqgP. We have identified the cleavage site that resides in the middle of the transmembrane domain of Spitz. Replacement of two residues that contribute to the scissile bond by Ala did not eliminate cleavage, but rather led to additional or alternative cleavages. Moreover, we have demonstrated that soluble YqgP exists as oligomers that are required for catalytic activity. These results suggest that soluble oligomers of maltose binding protein-YqgP complexes form micellelike structures that are able to retain the active conformation of the protease for catalysis. Therefore, this work not only provides a unique system for elucidating the reaction mechanism of rhomboid but also will facilitate the characterization of other intramembrane proteases as well as non-protease membrane proteins.     

Structural effect of a recombinant monoclonal antibody on hinge region peptide bond hydrolysis

IgG hinge region peptide bonds are susceptible to degradation by hydrolysis. To study the effect of Fab and Fc on hinge region peptide bond hydrolysis, a recombinant humanized monoclonal IgG1 antibody, its F(ab')2 fragment, and a model peptide with amino acid sequence corresponding to the hinge region were incubated at 40 degrees C in formulation buffer including complete protease inhibitor and EDTA for 0, 2, 4, 6 and 8 weeks. Two major cleavage sites were identified in the hinge region of the intact recombinant humanized monoclonal antibody and its F(ab')2 fragment, but only one major cleavage site of the model peptide was identified. Hinge region peptide bond hydrolysis of the intact antibody and its F(ab')2 fragment degraded at comparable rates, while the model peptide degraded much faster. It was concluded that Fab region of the IgG, but not Fc portion had significant effect on preventing peptide bond cleavage by direct hydrolysis. Hydrolysis of hinge region peptide bonds was accelerated under both acidic and basic conditions.     

A cleavage enzyme-cytometric bead array provides biochemical profiling of resistance mutations in HIV-1 Gag and protease

Most protease-substrate assays rely on short, synthetic peptide substrates consisting of native or modified cleavage sequences. These assays are inadequate for interrogating the contribution of native substrate structure distal to a cleavage site that influences enzymatic cleavage or for inhibitor screening of native substrates. Recent evidence from HIV-1 isolates obtained from individuals resistant to protease inhibitors has demonstrated that mutations distal to or surrounding the protease cleavage sites in the Gag substrate contribute to inhibitor resistance. We have developed a protease-substrate cleavage assay, termed the cleavage enzyme- cytometric bead array (CE-CBA), which relies on native domains of the Gag substrate containing embedded cleavage sites. The Gag substrate is expressed as a fluorescent reporter fusion protein, and substrate cleavage can be followed through the loss of fluorescence utilizing flow cytometry. The CE-CBA allows precise determination of alterations in protease catalytic efficiency (k(cat)/K(M)) imparted by protease inhibitor resistance mutations in protease and/or gag in cleavage or noncleavage site locations in the Gag substrate. We show that the CE-CBA platform can identify HIV-1 protease present in cellular extractions and facilitates the identification of small molecule inhibitors of protease or its substrate Gag. Moreover, the CE-CBA can be readily adapted to any enzyme-substrate pair and can be utilized to rapidly provide assessment of catalytic efficiency as well as systematically screen for inhibitors of enzymatic processing of substrate.     

A fluorescent oligopeptide energy transfer assay with broad applications for neutral proteases

A fluorescent peptide substrate to explore the protease specificity for the amino acid regions C- and N-terminal to the cleavage site has been designed. Intramolecular quenching of indole fluorescence by an N-terminal dansyl group separated by six amino acid residues forms the basis of this assay. For a particular enzyme, specificity can be designed into the peptide sequence by means of the number of residues that separate the two chromophores. In the present instance, the heptapeptide Dns-Gly-Lys-Tyr-Ala-Pro-Trp-Val is used to assay angiotensin converting enzyme (ACE), Astacus protease, carboxypeptidase A, alpha-chymotrypsin, and trypsin, all of which cleave the peptide in accord with their known specificity: Trypsin and Astacus protease hydrolyze only the Lys-Tyr and Tyr-Ala bonds, respectively. alpha-Chymotrypsin primarily cleaves the Tyr-Ala bond while ACE makes three successive dipeptidyl cleavages from the C-terminus. Carboxypeptidase rapidly hydrolyzes first the Trp-Val and then the Pro-Trp bond. For all of the enzymes, catalytic activity (kcat/Km) is in the range from 10(5) to 10(6) M-1 s-1. Hydrolysis causes a fluorescence increase in the 310 to 410 nm region of 8.6- to 13.6-fold depending on the enzyme that is assayed. Assays can be designed based on the increase in tryptophan fluorescence or by individual product analyses using thin-layer or high-performance liquid chromatography. The specificity and sensitivity of such internally quenched fluorescent oligopeptides would seem to be ideal for the assay of specific endoproteases.     

Maturation of the catalytic alpha-subunit of Na,K-ATPase during intracellular transport

The protease sensitivity of the catalytic alpha-subunit of Na,K-ATPase during intracellular transport along the exocytic pathway has been investigated in two amphibian epithelial cell lines. Controlled trypsinolysis followed by immunoprecipitation of cell homogenates or microsomal fractions from [35S]methionine pulse-chased A6 kidney cells revealed distinct cleavage patterns by SDS-PAGE. Shortly after synthesis (7-min pulse), the 98-kD alpha-subunit is fully sensitive to trypsin digestion and is cleaved into a 35-kD membrane-bound and a 27.5- kD soluble peptide. With a 15-min pulse, 10% of the newly synthesized polypeptide becomes resistant to trypsin digestion. With longer chase time, the proportion of protease-resistant alpha-subunit further increases. Concomitantly, the alpha-subunit acquires the ability to undergo cation-dependent conformational transitions, as reflected by distinct tryptic digest patterns in the presence of Na+ or K+. Similar results were obtained in TBM cells, a toad bladder cell line. Our data indicate that the catalytic subunit of Na,K-ATPase is structurally rearranged during intracellular transport from its site of synthesis to its site of action at the cell surface, a modification which might mark the functional maturation of the enzyme.     

Cargo sequences are important for Som1p-dependent signal peptide cleavage in yeast mitochondria

The inner membrane protease (IMP) has two catalytic subunits, Imp1p and Imp2p, that exhibit nonoverlapping substrate specificity in mitochondria of the yeast Saccharomyces cerevisiae. The IMP also has at least one noncatalytic subunit, Som1p, which is required to cleave signal peptides from a subset of Imp1p substrates. To understand how Som1p mediates Imp1p substrate specificity, we addressed the possibility that Som1p functions as a molecular chaperone, which binds to specific substrates and directs them to the catalytic site. Our results show that cargo sequences attached to the signal peptide are important for Som1p-dependent presequence cleavage; however, no specific cargo sequence is required. Indeed, we show that a substrate normally destined for Imp2p is cleaved in a Som1p-dependent manner when the substrate is directed to Imp1p. These results argue against the notion that Som1p is a molecular chaperone. Instead, we propose that the cargo of some Imp1p substrates can assume a conformation incompatible with presequence cleavage. Som1p could thus act through Imp1p to improve cleavage efficiency early during substrate maturation.     

Degradation of a signal peptide by protease IV and oligopeptidase A.

The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.     

The degradation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase into the 44-kDa fragment in the lysates of chloroplasts incubated in darkness

Lysates of chloroplasts isolated from naturally senescing wheat leaves were incubated in darkness. The 44-kDa fragment, lacking the N-terminal-side portion of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (LSU), was found by immunoblotting with the LSU site-specific antibodies. Analysis of its N-terminal amino acid sequence indicated that the LSU was specifically cleaved at the peptide bond between Phe-40 and Arg-41. The site was located on the surface of the molecule and faced outward. Such cleavage of the LSU has not been previously reported. It is indicated that the cleavage was triggered by an unknown protease existing in chloroplasts.     

Crystal structures of highly constrained substrate and hydrolysis products bound to HIV-1 protease. Implications for the catalytic mechanism

HIV-1 protease is a key target in treating HIV infection and AIDS, with 10 inhibitors used clinically. Here we used an unusual hexapeptide substrate, containing two macrocyclic tripeptides constrained to mimic a beta strand conformation, linked by a scissile peptide bond, to probe the structural mechanism of proteolysis. The substrate has been cocrystallized with catalytically active synthetic HIV-1 protease and an inactive isosteric (D25N) mutant, and three-dimensional structures were determined (1.60 A). The structure of the inactive HIVPR(D25N)/substrate complex shows an intact substrate molecule in a single orientation that perfectly mimics the binding of conventional peptide ligands of HIVPR. The structure of the active HIVPR/product complex shows two monocyclic hydrolysis products trapped in the active site, revealing two molecules of the N-terminal monocyclic product bound adjacent to one another, one molecule occupying the nonprime site, as expected, and the other monocycle binding in the prime site in the reverse orientation. The results suggest that both hydrolysis products are released from the active site upon cleavage and then rebind to the enzyme. These structures reveal that N-terminal binding of ligands is preferred, that the C-terminal site is more flexible, and that HIVPR can recognize substrate shape rather than just sequence alone. The product complex reveals three carboxylic acids in an almost planar orientation, indicating an unusual hexagonal homodromic complex between three carboxylic acids. The data presented herein regarding orientation of catalytic aspartates support the cleavage mechanism proposed by Northrop. The results imply strategies for design of inhibitors targeting the N-terminal side of the cleavage site or taking advantage of the flexibility in the protease domain that accommodates substrate/inhibitor segments C-terminal to the cleavage site.     

The primary structure and structural characteristics of Achromobacter lyticus protease I, a lysine-specific serine protease

The complete amino acid sequence of Achromobacter lyticus protease I (EC 3.4.21.50), which specifically hydrolyzes lysyl peptide bonds, has been established. This has been achieved by sequence analysis of the reduced and S-carboxymethylated protease and of peptides obtained by enzymatic digestion with Achromobacter protease I itself and Staphylococcus aureus V8 protease and by chemical cleavage with cyanogen bromide. The protease consists of 268 residues with three disulfide bonds, which have been assigned to Cys6-Cys216, Cys12-Cys80, and Cys36-Cys58. Comparison of the amino acid sequence of Achromobacter protease and other serine proteases of bacterial and mammalian origins has revealed that Achromobacter protease I is a mammalian-type serine protease of which the catalytic triad comprises His57, Asp113, and Ser194. It has also been shown that the protease has 9- and 26-residue extensions of the peptide chain at the N and C termini, respectively, and overall sequence homology is as low as 20% with bovine trypsin. The presence of a disulfide bridge between the N-terminal extension Cys6 and Cys216 close to the putative active site in the C-terminal region is thought to be responsible for the generation of maximal proteolytic function in the pH range 8.5-10.7 and enhanced stability to denaturation.