Matrix metalloproteinase activity inactivates the CXC chemokine stromal cell-derived factor-1

Chemokines provide directional cues for leukocyte migration and activation that are essential for normal leukocytic trafficking and for host responses during processes such as inflammation, infection, and cancer. Recently we reported that matrix metalloproteinases (MMPs) modulate the activity of the CC chemokine monocyte chemoattractant protein-3 by selective proteolysis to release the N-terminal tetrapeptide. Here we report the N-terminal processing, also at position 4-5, of the CXC chemokines stromal cell-derived factor (SDF)-1alpha and beta by MMP-2 (gelatinase A). Robustness of the MMP family for chemokine cleavage was revealed from identical cleavage site specificity of MMPs 1, 3, 9, 13, and 14 (MT1-MMP) toward SDF-1; selectivity was indicated by absence of cleavage by MMPs 7 and 8. Efficient cleavage of SDF-1alpha by MMP-2 is the result of a strong interaction with the MMP hemopexin C domain at an exosite that overlaps the monocyte chemoattractant protein-3 binding site. The association of SDF-1alpha with different glycosaminoglycans did not inhibit cleavage. MMP cleavage of SDF-1alpha resulted in loss of binding to its cognate receptor CXCR-4. This was reflected in a loss of chemoattractant activity for CD34(+) hematopoietic progenitor stem cells and pre-B cells, and unlike full-length SDF-1alpha, the MMP-cleaved chemokine was unable to block CXCR-4-dependent human immunodeficiency virus-1 infection of CD4(+) cells. These data suggest that MMPs may be important regulatory proteases in attenuating SDF-1 function and point to a deep convergence of two important networks, chemokines and MMPs, to regulate leukocytic activity in vivo.     

Molecular determinants of metalloproteinase substrate specificity: matrix metalloproteinase substrate binding domains,modules, and exosites

The function of ancillary domains and modules attached to the catalytic domain of mutidomain proteases, such as the matrix metalloproteinases (MMPs), are not well understood. The importance of discrete MMP substrate binding sites termed exosites on domains located outside the catalytic domain was first demonstrated for native collagenolysis. The essential role of hemopexin carboxyl-domain exosites in the cleavage of noncollagenous substrates such as chemokines has also been recently revealed. This article updates a previous review of the role of substrate recognition by MMP exosites in both preparing complex substrates, such as collagen, for cleavage and for tethering noncollagenous substrates to MMPs for more efficient proteolysis. Exosite domain interaction and movements—“molecular tectonics”—that are required for native collagen triple helicase activity are discussed. The potential role of collagen binding in regulating MMP-2 (gelatinase A) activation at the cell surface reveals unexpected consequences of substrate interactions that can lead to collagen cleavage and regulation of the activation and activity of downstream proteinases necessary to complete the collagenolytic cascade.     

Identifi cation of protease exosite-interacting peptides that enhance substrate cleavage kinetics

Abstract Many peptidases are thought to require non-active site interaction surfaces, or exosites, to recognize and cleave physiological substrates with high specifi city and catalytic effi ciency. However, the existence and function of protease exosites remain obscure owing to a lack of effective methods to identify and characterize exosite-interacting substrates. To address this need, we modifi ed the cellular libraries of peptide substrates (CLiPS) methodology to enable the discovery of exosite-interacting peptide ligands. Invariant cleavage motifs recognized by the active sites of thrombin and caspase-7 were displayed on the outer surface of bacteria adjacent to a candidate exosite-interacting peptide. Exosite peptide libraries were then screened for ligands that accelerate cleavage of the active site recognition motif using two-color fl ow cytometry. Exosite CLiPS (eCLiPS) identifi ed exosite-binding peptides for thrombin that were highly similar to a critical exosite interaction motif in the thrombin substrate, proteaseactivated receptor 1. Protease activity probes incorporating exosite-binding peptides were cleaved ten-fold faster than substrates without exosite ligands, increasing their sensitivity to thrombin activity in vitro. For comparison, screening with caspase-7 yielded peptides that modestly enhanced (two-fold) substrate cleavage rates. The eCLiPS method provides a new tool to profi le the ligand specifi city of protease exosites and to develop improved substrates.     

The role of thrombin exosites I and II in the activation of human coagulation factor V

Human blood coagulation Factor V (FV) is a plasma protein with little procoagulant activity. Limited proteolysis at Arg(709), Arg(1018), and Arg(1545) by thrombin or Factor Xa (FXa) results in the generation of activated FV, which serves as a cofactor of FXa in prothrombin activation. Both thrombin exosites I and II have been reported to be involved in FV activation, but the relative importance of these regions in the individual cleavages remains unclear. To investigate the role of each exosite in FV activation, we have used recombinant FV molecules with only one of the three activation cleavage sites available, in combination with exosite I- or II-specific aptamers. In addition, structural requirements for exosite interactions located in the B-domain of FV were probed using FV B-domain deletion mutants and comparison with FV activating enzymes from the venom of Russell's viper (RVV-V) and of Levant's viper (LVV-V) known to activate FV by specific cleavage at Arg(1545). Our results indicate that thrombin exosite II is not involved in cleavage at Arg(709) and that both thrombin exosites are important for recognition and cleavage at Arg(1545). Efficient thrombin-catalyzed FV activation requires both the N- and C-terminal regions of the B-domain, whereas only the latter is required by RVV-V and LVV-V. This indicates that proteolysis of FV by thrombin at Arg(709), Arg(1018), and Arg(1545) show different cleavage requirements with respect to interactions mediated by thrombin exosites and areas that surround the respective cleavage sites. In addition, interactions between exosite I of thrombin and FV are primarily responsible for the different cleavage site specificity as compared with activation by RVV-V or LVV-V.     

Substrate recognition mechanism of VAMP/synaptobrevin-cleaving clostridial neurotoxins

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide bond in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle-associated membrane protein (VAMP)/synaptobrevin. TeNT and BoNT/B, D, F, and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/synaptobrevin. Except for BoNT/B and TeNT, they cleave unique peptide bonds, and prior work suggested that different substrate segments are required for the interaction of each toxin. Although the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, and F and TeNT by means of systematic mutagenesis of VAMP/synaptobrevin. For each of the toxins, three or more residues clustered at an N-terminal site remote from the respective scissile bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the scissile peptide bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4') do not play a role in the catalytic process. Mutation of residues in the proximity of the scissile bond exclusively affects the turnover number; however, the importance of individual positions at the cleavage sites varied for each toxin. The data show that, similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/synaptobrevin-specific clostridial neurotoxins also initiate substrate interaction, employing an exosite located N-terminal of the scissile peptide bond.     

Remote exosites of the catalytic domain of matrix metalloproteinase-12 enhance elastin degradation

How does matrix metalloproteinase-12 (MMP-12 or metalloelastase) degrade elastin with high specific activity? Nuclear magnetic resonance suggested soluble elastin covers surfaces of MMP-12 far from its active site. Two of these surfaces have been found, by mutagenesis guided by the BINDSIght approach, to affect degradation and affinity for elastin substrates but not a small peptide substrate. Main exosite 1 has been extended to Asp124 that binds calcium. Novel exosite 2 comprises residues from the II-III loop and β-strand I near the back of the catalytic domain. The high degree of exposure of these distal exosites may make them accessible to elastin made more flexible by partial hydrolysis. Importantly, the combination of one lesion each at exosites 1 and 2 and the active site decreased the catalytic competence toward soluble elastin by 13-18-fold to the level of MMP-3, homologue and poor elastase. Double-mutant cycle analysis of conservative mutations of Met156 (exosite 2) and either Asp124 (exosite 1) or Ile180 (active site) showed they had additive effects. Compared to polar substitutions observed in other MMPs, Met156 enhanced affinity and Ile180 the k(cat) for soluble elastin. Both residues detracted from the higher folding stability with polar mutations. This resembles the trend in enzymes of an inverse relationship between folding stability and activity. Restoring Asp124 from combination mutants enhanced the k(cat) for soluble elastin. In elastin degradation, exosites 1 and 2 contributed in a manner independent of each other and Ile180 at the active site, but with partial coupling to Ala182 near the active site. The concept of weak, separated interactions coalescing somewhat independently can be extended to this proteolytic digestion of a protein from fibrils.     

Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX

Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions.     

Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin

Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin''s active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity to its proteolytic activity by mediating binding to substrates, receptors, and cofactors. Exosite 1 mediates binding and cleavage of fibrinogen, proteolytically activated receptors, and some coagulation factors, while exosite 2 mediates binding to heparin and to platelet receptor GPIb-IX-V. The crystal structures of two nucleic acid ligands bound to thrombin have been solved. Previously Padmanabhan and colleagues solved the structure of a DNA aptamer bound to exosite 1 and we reported the structure of an RNA aptamer bound to exosite 2 on thrombin. Based upon these structural studies we speculated that the two aptamers would not compete for binding to thrombin. We observe that simultaneously blocking both exosites with the aptamers leads to synergistic inhibition of thrombin-dependent platelet activation and procoagulant activity. This combination of exosite 1 and exosite 2 inhibitors may provide a particularly effective antithrombotic approach.     

Elastin Degradation by Cathepsin V Requires Two Exosites

Cathepsin V is a highly effective elastase and has been implicated in physiological and pathological extracellular matrix degradation. However, its mechanism of action remains elusive. Whereas human cathepsin V exhibits a potent elastolytic activity, the structurally homologous cathepsin L, which shares a 78% amino acid sequence, has only a minimal proteolytic activity toward insoluble elastin. This suggests that there are distinct structural domains that play an important role in elastinolysis. In this study, a total of 11 chimeras of cathepsins V and L were generated to identify elastin-binding domains in cathepsin V. Evaluation of these chimeras revealed two exosites contributing to the elastolytic activity of cathepsin V that are distant from the active cleft of the protease and are located in surface loop regions. Replacement of exosite 1 or 2 with analogous residues from cathepsin L led to a 75 and 43% loss in the elastolytic activity, respectively. Replacement of both exosites yielded a non-elastase variant similar to that of cathepsin L. Identification of these exosites may contribute to the design of inhibitors that will only affect the elastolytic activity of cysteine cathepsins without interfering with other physiological protease functions.     

Regulation of the catalytic function of coagulation factor VIIa by a conformational linkage of surface residue Glu 154 to the active site

Coagulation factor VIIa is an allosterically regulated trypsin-like serine protease that initiates the coagulation pathways upon complex formation with its cellular receptor and cofactor tissue factor (TF). The analysis of a conformation-sensitive monoclonal antibody directed to the macromolecular substrate exosite in the VIIa protease domain demonstrated a conformational link from this exosite to the catalytic cleft that is independent of cofactor-induced allosteric changes. In this study, we identify Glu 154 as a critical surface-exposed exosite residue side chain that undergoes conformational changes upon active site inhibitor binding. The Glu 154 side chain is important for hydrolysis of scissile bond mimicking peptidyl p-nitroanilide substrates, and for inhibition of VIIa's amidolytic function upon antibody binding. This exosite residue is not linked to the catalytic cleft residue Lys 192 which plays an important role in thrombin's allosteric coupling to exosite I. Allosteric linkages between VIIa's active site and the cofactor binding site or between the cofactor binding site and the macromolecular substrate exosite were not influenced by mutation of Glu 154. Glu 154 thus only influences the linkage of the macromolecular substrate binding exosite to the catalytic center. These data provide novel evidence that allosteric regulation of VIIa's catalytic function involves discrete and independent conformational linkages and that allosteric transitions in the VIIa protease domain are not globally coupled.     

Mechanisms of Gasdermin Recognition by Proteases

Members of the gasdermin family contain positively charged N-terminal domains (NTDs) capable of binding phospholipids and assembling membrane pores, and C-terminal domains (CTDs) that bind the NTDs to prevent pore formation in the resting states. The flexible NTD-CTD linker regions of gasdermins are highly variable in length and sequences, which may be attributable to gasdermin recognition by diverse proteases. In addition, protease cleavage within the NTDs is known to inactivate several gasdermin family members. Recognition and cleavage of the gasdermin family members by different proteases share common and distinct features at the protease active sites, as well as exosites recently identified for the inflammatory caspases. Utilization of exosites may strengthen enzyme-substrate interaction, improve efficiency of proteolysis, and enhance substrate selectivity. It remains to be determined if the dual site recognition of gasdermin D (GSDMD) by the inflammatory caspases is employed by other GSDMD-targeting proteases, or is involved in proteolytic processing of other gasdermins. Biochemical and structural approaches will be instrumental in revealing how potential exosites in diverse proteases engage different gasdermin substrates. Different features of gasdermin sequence, structure, expression characteristics, and post-translational modifications may dictate distinct mechanisms of protease-dependent activation or inactivation. Such diverse mechanisms may underlie the divergent physiological and pathological functions of gasdermins, and furnish opportunities for therapeutic targeting of gasdermins in infectious diseases and inflammatory disorders.     

Metadegradomics: toward in vivo quantitative degradomics of proteolytic post-translational modifications of the cancer proteome

Post-translational modifications enable extra layers of control of the proteome, and perhaps the most important is proteolysis, a major irreversible modification affecting every protein. The intersection of the protease web with a proteome sculpts that proteome, dynamically modifying its state and function. Protease expression is distorted in cancer, so perturbing signaling pathways and the secretome of the tumor and reactive stromal cells. Indeed many cancer biomarkers are stable proteolytic fragments. It is crucial to determine which proteases contribute to the pathology versus their roles in homeostasis and in mitigating cancer. Thus the full substrate repertoire of a protease, termed the substrate degradome, must be deciphered to define protease function and to identify drug targets. Degradomics has been used to identify many substrates of matrix metalloproteinases that are important proteases in cancer. Here we review recent degradomics technologies that allow for the broadly applicable identification and quantification of proteases (the protease degradome) and their activity state, substrates, and interactors. Quantitative proteomics using stable isotope labeling, such as ICAT, isobaric tags for relative and absolute quantification (iTRAQ), and stable isotope labeling by amino acids in cell culture (SILAC), can reveal protease substrates by taking advantage of the natural compartmentalization of membrane proteins that are shed into the extracellular space. Identifying the actual cleavage sites in a complex proteome relies on positional proteomics and utilizes selection strategies to enrich for protease-generated neo-N termini of proteins. In so doing, important functional information is generated. Finally protease substrates and interactors can be identified by interactomics based on affinity purification of protease complexes using exosite scanning and inactive catalytic domain capture strategies followed by mass spectrometry analysis. At the global level, the N terminome analysis of whole communities of proteases in tissues and organs in vivo provides a full scale understanding of the protease web and the web-sculpted proteome, so defining metadegradomics.     

Macromolecular assembly-driven processing of the 2/3 cleavage site in the alphavirus replicase polyprotein

Semliki Forest virus (SFV) is a member of the Alphavirus genus, which produces its replicase proteins in the form of a nonstructural (ns) polyprotein precursor P1234. The maturation of the replicase occurs in a temporally controlled manner by protease activity of nsP2. The template preference and enzymatic capabilities of the alphaviral replication complex have a very important connection with its composition, which is irreversibly altered by proteolysis. The final cleavage of the 2/3 site in the ns polyprotein apparently leads to significant rearrangements within the replication complex and thus denotes the "point of no return" for viral replication progression. Numerous studies have devised rules for when and how ns protease acts, but how the alphaviral 2/3 site is recognized remained largely unexplained. In contrast to the other two cleavage sites within the ns polyprotein, the 2/3 site evidently lacks primary sequence elements in the vicinity of the scissile bond sufficient for specific protease recognition. In this study, we sought to investigate the molecular details of the regulation of the 2/3 site processing in the SFV ns polyprotein. We present evidence that correct macromolecular assembly, presumably strengthened by exosite interactions rather than the functionality of the individual nsP2 protease, is the driving force for specific substrate targeting. We conclude that structural elements within the macrodomain of nsP3 are used for precise positioning of a substrate recognition sequence at the catalytic center of the protease and that this process is coordinated by the exact N-terminal end of nsP2, thus representing a unique regulation mechanism used by alphaviruses.     

Direct Visualization of Protease Action on Collagen Triple Helical Structure

Enzymatic processing of extracellular matrix (ECM) macromolecules by matrix metalloproteases (MMPs) is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen ¾ fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease.     

Macromolecular substrate-binding exosites on both the heavy and light chains of factor XIa mediate the formation of the Michaelis complex required for factor IX-activation

Binding of factor IX (FIX) to an exosite on the heavy chain of factor XIa (FXIa) is essential for the optimal activation of FIX (Sinha, D., Seaman, F. S., and Walsh, P. N. (1987) Biochemistry 26, 3768-3775). To gain further insight into the mechanisms of activation of FIX by FXIa, we have investigated the kinetic properties of FXIa-light chain (FXIa-LC) with its active site occupied by either a reversible inhibitor of serine proteases (p-aminobenzamidine, PAB) or a small peptidyl substrate (S-2366) and have examined FIX cleavage products resulting from activation by FXIa or FXIa-LC. PAB inhibited the hydrolysis of S-2366 by FXIa-LC in a classically competitive fashion. In contrast, PAB was found to be a noncompetitive inhibitor of the activation of the macromolecular substrate FIX. Occupancy of the active site of the FXIa-LC by S-2366 also resulted in noncompetitive inhibition of FIX activation. These results demonstrate the presence of an exosite for FIX binding on the FXIa-LC remote from its active site. Furthermore, examination of the cleavage products of FIX indicated that in the absence of either Ca2+ or the heavy chain of FXIa there was substantial accumulation of the inactive intermediate FIXalpha, indicating a slower rate of cleavage of the scissile bond Arg180-Val181. We conclude that binding to two substrate-binding exosites one on the heavy chain and the other on the light chain of FXIa is required to mediate the formation of the Michaelis complex and efficient cleavages of the two spatially separated scissile bonds of FIX.     

Proteomic approaches to uncover MMP function

Proteomics has revolutionized protease research and particularly contributed to the identification of novel substrates and their sites of cleavage as key determinants of protease function. New technologies and rapid advancements in development of powerful mass spectrometers allowed unprecedented insights into activities of matrix metalloproteinases (MMPs) within their complex extracellular environments. Mass spectrometry-based proteomics extended our knowledge on MMP cleavage specificities and will help to develop more specific inhibitors as new therapeutics. Quantitative proteomics and N-terminal enrichment strategies have revealed numerous novel MMP substrates and shed light on their modes of action in vitro and in vivo. In this review, we provide an overview of current proteomic technologies in protease research and their application to the functional characterization of MMPs.     

Conformational changes in thrombin when complexed by serpins

Thrombin possesses two positively charged surface domains, termed exosites, that orient substrates and inhibitors for reaction with the enzyme. Because the exosites also allosterically modulate thrombin's activity, we set out to determine whether the structure or function of the exosites changes when thrombin forms complexes with antithrombin, heparin cofactor II, or alpha(1)-antitrypsin (M358R), serpins that utilize both, one, or neither of the exosites, respectively. Using a hirudin-derived peptide to probe the integrity of exosite 1, no binding was detected when thrombin was complexed with heparin cofactor II or alpha(1)-antitrypsin (M358R), and the peptide exhibited a 55-fold lower affinity for the thrombin-antithrombin complex than for thrombin. Bound peptide or HD-1, an exosite 1-binding DNA aptamer, was displaced from thrombin by each of the three serpins. Thrombin binding to fibrin also was abrogated when the enzyme was complexed with serpins. These data reveal that, regardless of the initial mode of interaction, the function of exosite 1 is lost when thrombin is complexed by serpins. In contrast, the integrity of exosite 2 is largely retained when thrombin is complexed by serpins, because interaction with heparin or an exosite 2-directed DNA aptamer was only modestly altered. The disorganization of exosite 1 that occurs when thrombin is complexed by serpins is consistent with results of protease sensitivity studies and crystallographic analysis of a homologous enzyme-serpin complex.     

GpIbα Interacts Exclusively with Exosite II of Thrombin

Activation of platelets by the serine protease thrombin is a critical event in haemostasis. This process involves the binding of thrombin to glycoprotein Ibα (GpIbα) and cleavage of protease-activated receptors (PARs). The N-terminal extracellular domain of GpIbα contains an acidic peptide stretch that has been identified as the main thrombin binding site, and both anion binding exosites of thrombin have been implicated in GpIbα binding, but it remains unclear how they are involved. This issue is of critical importance for the mechanism of platelet activation by thrombin. If both exosites bind to GpIbα, thrombin could potentially act as a platelet adhesion molecule or receptor dimerisation trigger. Alternatively, if only a single site is involved, GpIbα may serve as a cofactor for PAR-1 activation by thrombin. To determine the involvement of thrombin's two exosites in GpIbα binding, we employed the complementary methods of mutational analysis, binding studies, X-ray crystallography and NMR spectroscopy. Our results indicate that the peptide corresponding to the C-terminal portion of GpIbα and the entire extracellular domain bind exclusively to thrombin's exosite II. The interaction of thrombin with GpIbα thus serves to recruit thrombin activity to the platelet surface while leaving exosite I free for PAR-1 recognition.     

Identification of the substrate-binding exosites of two snake venom serine proteinases: molecular basis for the partition of two essential functions of thrombin

The venom of the South American snake Bothrops jararaca contains two serine proteinases, bothrombin and the platelet-aggregating enzyme PA-BJ, which share 66% sequence identity. Each of these proteinases possesses one of the two essential procoagulant functions of thrombin-the clotting of fibrinogen and platelet aggregation. Thus, bothrombin clots fibrinogen but has no direct effect on platelets, unless in the presence of exogenous fibrinogen. PA-BJ induces platelet aggregation by interacting with the protease-activated platelet receptor without clotting fibrinogen. On the other hand, thrombin possesses two extended surfaces. One is composed of basic and hydrophobic residues (exosite I) and the other one of basic residues only (exosite II). These exosites are involved in the recognition of physiological macromolecular substrates. In order to identify the corresponding exosites in bothrombin and PA-BJ and understand the molecular basis of the partition of the two procoagulant functions of thrombin among the two snake venom enzymes, we used molecular modeling to obtain models of their complexes with their natural substrates fibrinogen and a fragment of the protease-activated platelet receptor, respectively. In analogy to thrombin, each of the enzymes presents two exosites. Nonetheless, the exosites contain a smaller proportion of basic residues than thrombin does (45-72%), reducing thus the functional diversity of the enzymes. In addition, the composition of exosite I is different in both enzymes. We identify those residues in exosite I that could contribute to the differences in specificity. Finally, allostery does not seem to mediate macromolecular substrate recognition by these enzymes.     

Peptide substrates and inhibitors of the HIV-1 protease

Oligopeptides containing the consensus retroviral protease cleavage sequence Ser/Thr-X-Y-Tyr/Phe-Pro are substrates for purified recombinant HIV-1 protease with Km's in the millimolar range. The minimum sequence containing the consensus pentapeptide which serves as a good substrate is a heptapeptide spanning the P4-P3' residues. Substitution of reduced Phe-Pro or Tyr-Pro dipeptide isosteres or the statine analog 3-hydroxy-4-amino-5-phenylpentanoic acid for the scissile dipeptide afforded inhibitors of HIV-1 protease with Ki values in the micromolar range, three orders of magnitude better in affinity than the corresponding substrates. Inhibitors of HIV-1 protease may provide a novel and potentially useful therapeutic approach to the treatment of acquired immune deficiency syndrome (AIDS).