S1 Specificity Pocket Research Articles

Structural and time-resolved mechanistic investigations of protein hydrolysis by the acidic proline-specific endoprotease from Aspergillus niger.

Proline-specific endoproteases have been successfully used in, for example, the in-situ degradation of gluten, the hydrolysis of bitter peptides, the reduction of haze during beer production, and the generation of peptides for mass spectroscopy and proteomics applications. Here we present the crystal structure of the extracellular proline-specific endoprotease from Aspergillus niger (AnPEP), a member of the S28 peptidase family with rarely observed true proline-specific endoprotease activity. Family S28 proteases have a conventional Ser-Asp-His catalytic triad, but their oxyanion-stabilizing hole shows a glutamic acid, an amino acid not previously observed in this role. Since these enzymes have an acidic pH optimum, the presence of a glutamic acid in the oxyanion hole may confine their activity to an acidic pH. Yet, considering the presence of the conventional catalytic triad, it is remarkable that the A. niger enzyme remains active down to pH 1.5. The determination of the primary cleavage site of cytochrome c along with molecular dynamics-assisted docking studies indicate that the active site pocket of AnPEP can accommodate a reverse turn of approximately 12 amino acids with proline at the S1 specificity pocket. Comparison with the structures of two S28-proline-specific exopeptidases reveals not only a more spacious active site cavity but also the absence of any putative binding sites for amino- and carboxyl-terminal residues as observed in the exopeptidases, explaining AnPEP's observed endoprotease activity.

SARS-CoV-2 host cell entry: an in silico investigation of potential inhibitory roles of terpenoids

BackgroundTargeting viral cell entry proteins is an emerging therapeutic strategy for inhibiting the first stage of SARS-CoV-2 infection. In this study, 106 bioactive terpenoids from African medicinal plants were screened through molecular docking analysis against human angiotensin-converting enzyme 2 (hACE2), human transmembrane protease serine 2 (TMPRSS2), and the spike (S) proteins of SARS-CoV-2, SARS-CoV, and MERS-CoV. In silico absorption-distribution-metabolism-excretion-toxicity (ADMET) and drug-likeness prediction, molecular dynamics (MD) simulation, binding free energy calculations, and clustering analysis of MD simulation trajectories were performed on the top docked terpenoids to respective protein targets. ResultsThe results revealed eight terpenoids with high binding tendencies to the catalytic residues of different targets. Two pentacyclic terpenoids (24-methylene cycloartenol and isoiguesteri) interacted with the hACE2 binding hotspots for the SARS-CoV-2 spike protein, while the abietane diterpenes were found accommodated within the S1-specificity pocket, interacting strongly with the active site residues TMPRSS2. 3-benzoylhosloppone and cucurbitacin interacted with the RBD and S2 subunit of SARS-CoV-2 spike protein respectively. These interactions were preserved in a simulated dynamic environment, thereby, demonstrating high structural stability. The MM-GBSA binding free energy calculations corroborated the docking interactions. The top docked terpenoids showed favorable drug-likeness and ADMET properties over a wide range of molecular descriptors. ConclusionThe identified terpenoids from this study provides core structure that can be exploited for further lead optimization to design drugs against SARS-CoV-2 cell-mediated entry proteins. They are therefore recommended for further in vitro and in vivo studies towards developing entry inhibitors against the ongoing COVID-19 pandemic.

Structural insights unravel the zymogenic mechanism of the virulence factor gingipain K from Porphyromonas gingivalis, a causative agent of gum disease from the human oral microbiome

Skewing of the human oral microbiome causes dysbiosis and preponderance of bacteria such as Porphyromonas gingivalis, the main etiological agent of periodontitis. P. gingivalis secretes proteolytic gingipains (Kgp and RgpA/B) as zymogens inhibited by a pro-domain that is removed during extracellular activation. Unraveling the molecular mechanism of Kgp zymogenicity is essential to design inhibitors blocking its activity. Here, we found that the isolated 209-residue Kgp pro-domain is a boomerang-shaped all-β protein similar to the RgpB pro-domain. Using composite structural information of Kgp and RgpB, we derived a plausible homology model and mechanism of Kgp-regulating zymogenicity. Accordingly, the pro-domain would laterally attach to the catalytic moiety in Kgp and block the active site through an exposed inhibitory loop. This loop features a lysine (Lys129) likely occupying the S1 specificity pocket and exerting latency. Lys129 mutation to glutamate or arginine led to misfolded protein that was degraded in vivo Mutation to alanine gave milder effects but still strongly diminished proteolytic activity, without affecting the subcellular location of the enzyme. Accordingly, the interactions of Lys129 within the S1 pocket are also essential for correct folding. Uniquely for gingipains, the isolated Kgp pro-domain dimerized through an interface, which partially overlapped with that between the catalytic moiety and the pro-domain within the zymogen, i.e. both complexes are mutually exclusive. Thus, pro-domain dimerization, together with partial rearrangement of the active site upon activation, explains the lack of inhibition of the pro-domain in trans. Our results reveal that the specific latency mechanism of Kgp differs from those of Rgps.

Distinctive binding modes and inhibitory mechanisms of two peptidic inhibitors of urokinase-type plasminogen activator with isomeric P1 residues

Two isomeric piperidine derivatives (meta and para isomers) were used as arginine mimics in the P1 position of a cyclic peptidic inhibitor (CPAYSRYLDC) of urokinase-type plasminogen activator. The two resulting cyclic peptides showed vastly different affinities (∼70 fold) to the target enzyme. X-ray crystal structure analysis showed that the two P1 residues were inserted into the S1 specificity pocket in indistinguishable manners. However, the rest of the peptides bound in entirely different ways on the surface of the enzyme, and the two peptides have different conformations, despite the highly similar sequence. These results demonstrate how the subtle difference in P1 residue can dictate the exosite interactions and the potencies of peptidic inhibitors, and highlight the importance of the P1 residue for protease inhibition. This study provides important information for the development of peptidic agents for pharmacological intervention.

A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility.

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.

Allosteric Inactivation of a Trypsin-Like Serine Protease by An Antibody Binding to the 37- and 70-Loops

Serine protease catalytic activity is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. Inhibitory monoclonal antibodies binding to such exosites are potential therapeutics and offer opportunities for elucidating fundamental allosteric mechanisms. The monoclonal antibody mU1 has previously been shown to be able to inhibit the function of murine urokinase-type plasminogen activator in vivo. We have now mapped the epitope of mU1 to the catalytic domain's 37- and 70-loops, situated about 20 Å from the S1 specificity pocket of the active site. Our data suggest that binding of mU1 destabilizes the catalytic domain and results in conformational transition into a state, in which the N-terminal amino group of Ile16 is less efficiently stabilizing the oxyanion hole and in which the active site has a reduced affinity for substrates and inhibitors. Furthermore, we found evidence for functional interactions between residues in uPA's C-terminal catalytic domain and its N-terminal A-chain, as deletion of the A-chain facilitates the mU1-induced conformational distortion. The inactive, distorted state is by several criteria similar to the E* conformation described for other serine proteases. Hence, agents targeting serine protease conformation through binding to exosites in the 37- and 70-loops represent a new class of potential therapeutics.

A Naturally Variable Residue in the S1 Subsite of M1 Family Aminopeptidases Modulates Catalytic Properties and Promotes Functional Specialization

M1 family metallo-aminopeptidases fulfill a wide range of critical and in some cases medically relevant roles in humans and human pathogens. The specificity of M1-aminopeptidases is dominated by the interaction of the well defined S1 subsite with the side chain of the first (P1) residue of the substrate and can vary widely. Extensive natural variation occurs at one of the residues that contributes to formation of the cylindrical S1 subsite. We investigated whether this natural variation contributes to diversity in S1 subsite specificity. Effects of 11 substitutions of the S1 subsite residue valine 459 in the Plasmodium falciparum aminopeptidase PfA-M1 and of three substitutions of the homologous residue methionine 260 in Escherichia coli aminopeptidase N were characterized. Many of these substitutions altered steady-state kinetic parameters for dipeptide hydrolysis and remodeled S1 subsite specificity. The most dramatic change in specificity resulted from substitution with proline, which collapsed S1 subsite specificity such that only substrates with P1-Arg, -Lys, or -Met were appreciably hydrolyzed. The structure of PfA-M1 V459P revealed that the proline substitution induced a local conformational change in the polypeptide backbone that resulted in a narrowed S1 subsite. The restricted specificity and active site backbone conformation of PfA-M1 V459P mirrored those of endoplasmic reticulum aminopeptidase 2, a human enzyme with proline in the variable S1 subsite position. Our results provide compelling evidence that changes in the variable residue in the S1 subsite of M1-aminopeptidases have facilitated the evolution of new specificities and ultimately novel functions for this important class of enzymes.

Interconversion of Active and Inactive Conformations of Urokinase-Type Plasminogen Activator

The catalytic activity of serine proteases depends on a salt-bridge between the amino group of residue 16 and the side chain of Asp194. The salt-bridge stabilizes the oxyanion hole and the S1 specificity pocket of the protease. Some serine proteases exist in only partially active forms, in which the amino group of residue 16 is exposed to the solvent. Such a partially active state is assumed by a truncated form of the murine urokinase-type plasminogen activator (muPA), consisting of residues 16-243. Here we investigated the allosteric interconversion between partially active states and the fully active state. Both a monoclonal antibody (mU3) and a peptidic inhibitor (mupain-1--16) stabilize the active state. The epitope of mU3 is located in the 37- and 70-loops at a site homologous to exosite I of thrombin. The N-terminus((Ile16)) of muPA((16--243)) was less exposed upon binding of mU3 or mupain-1--16. In contrast, introduction of the mutations F40Y or E137A into muPA((16--243)) increased exposure of the N-terminus((Ile16)) and resulted in large changes in the thermodynamic parameters for mupain-1--16 binding. We conclude that the distorted state of muPA((16--243)) is conformationally ordered upon binding of ligands to the active site and upon binding of mU3 to the 37- and 70-loops. Our study establishes the 37- and 70-loops as a unique site for binding to compounds stabilizing the active state of serine proteases.

Impaired bortezomib binding to mutant β5 subunit of the proteasome is the underlying basis for bortezomib resistance in leukemia cells

Proteasome inhibition is a novel treatment for several hematological malignancies. However, resistance to the proteasome inhibitor bortezomib (BTZ, Velcade) is an emerging clinical impediment. Mutations in the β5 subunit of the proteasome, the primary target of BTZ, have been associated with drug resistance. However, the exact mechanism by which these mutations contribute to BTZ resistance, is still largely unknown. Toward this end, we here developed BTZ-resistant multiple myeloma (8226) and acute lymphoblastic leukemia (CCRF-CEM) cell line models by exposure to stepwise increasing concentrations of BTZ. Characterization of the various BTZ-resistant cells revealed upregulation of mutant β5 subunit of the proteasome. These newly identified β5-subunit mutations, along with previously described mutations, formed a mutation cluster region in the BTZ-binding pocket of the β5 subunit, that of the S1 specificity pocket in particular. Moreover, we provide the first evidence that the mechanism underlying BTZ resistance in these tumor cells is impaired binding of BTZ to the mutant β5 subunit of the proteasome. We propose that proteasome subunit overexpression is an essential compensatory mechanism for the impaired catalytic activity of these mutant proteasomes. Our findings further suggest that second-generation proteasome inhibitors that target the α7 subunit of the proteasome can overcome this drug resistance modality.

Binding of alkaloids into the S1 specificity pocket of α-chymotrypsin: Evidence from induced circular dichroism spectra

Non-covalent binding of planar aromatic molecules into the S1 specificity pocket of the serine protease α-chymotrypsin (αCHT) can be detected by measuring induced circular dichroism (CD) spectroscopic signals. Utilizing this phenomenon, αCHT association of proflavine (PRF), the well known serine protease inhibitor has been investigated together with plant-derived compounds including isoquinoline, pyridocarbazole and indoloquinoline alkaloids, of which αCHT binding has never been reported. Non-degenerate exciton coupling between π-π* transitions of the ligand molecules and two tryptophan residues (Trp172 and Trp215) near to the binding site is proposed to be responsible for the induced CD activity. The association constants calculated from CD titration data indicated strong αCHT association of sanguninarine, ellipticine, desmethyl-isocryptolepine and isoneocryptolepine (K(a) ≈ 10(5) M(-1)) while berberine, coptisine and chelerythrine bind to the enzyme with lower, PRF-like affinity (K(a) ≈ 10(4) M(-1)). PRF-trypsin and ellipticine-trypsin binding interactions have also been demonstrated. The binding of the alkaloids into the S1 pocket of αCHT has been confirmed by CD competition experiments. Molecular docking calculations showed the inclusion of PRF as well as the alkaloid molecules in the S1 cavity where they are stabilized by hydrophobic and H-bonding interactions. These novel nonpeptidic scaffolds can be used for developing selective inhibitors of serine proteases having chymotrypsin-like folds. Furthermore, the results provide a novel, CD spectroscopic based approach for probing the ligand binding of αCHT and related proteases.

Structural and Mutational Analysis of Canonical Loop Residues In Protease Nexin 2 Involved In Factor XIa and Trypsin Inhibition.

AbstractAbstract 1147Factor XIa (FXIa) activates FIX and is regulated by platelet-secreted protease nexin 2 (PN2) that contains a Kunitz-type protease inhibitor (KPI) domain. Trypsin is regulated by basic pancreatic trypsin inhibitor (BPTI). The primary and tertiary structures of trypsin and the catalytic domain of FXIa are highly homologous, and KPI and BPTI are nearly identical structurally. We have previously identified two loop structures (loops 1 and 2) in the KPI domain of PN2 that interact with residues in the FXIa catalytic domain. Based on the structure of the FXIa/KPI complex crystal structure, residues within loops 1 and 2 were mutated for experiments examining the inhibition of FXIa and trypsin.Results show that the loop-1-region P1 site residue Arg15 of PN2KPI plays a major role in FXIa inhibition by protruding into the S1 specificity pocket of FXIa. Ala mutation at this site renders PN2KPI non-inhibitory for both FXIa and trypsin. BPTI has Lys15 at the P1 site. BPTI inhibits both FXIa and trypsin significantly less effectively than PN2KPI. PN2KPI-R15K lost FXIa inhibitory activity, whereas BPTI-K15R substantially gained affinity for FXIa. Like FXIa, trypsin preferred BPTI-K15R showing a significant enhancement in affinity. Thus, a major determinant of the inhibitory activity of PN2KPI and BPTI against FXIa and trypsin is the P1 residue, with Arg being preferred over Lys for both inhibitors and both proteases. In addition, loop 1 residues Pro13 and Arg20 make important contributions to both FXIa and trypsin inhibition as demonstrated by significantly elevated Ki values for Ala mutations (P13A, and R20A) at these sites. In contrast, Ser19 makes no significant contribution to inhibition of either FXIa or trypsin whereas Met17 makes a significant contribution to the inhibition of trypsin, but not FXIa. In loop 2, only Phe34 is identified as a residue making significant contributions to the inhibition of both FXIa and trypsin, since the PN2KPI-F34A mutant displayed reduced inhibitory activity for both FXIa (6-fold) and for trypsin (3-fold). To rationalize these findings, we examined the crystal structures of the FXIa(catalytic domain)/PN2KPI complex, and the trypsin/BPTI complex. Structurally, the PN2KPI loop-1, P1-site residue Arg15 makes a complex primary interaction with Asp189 of both FXIa and trypsin. Disruption of this site by R15A mutation renders PN2KPI non-inhibitory because it preempts salt bridge interactions from two nitrogen atoms of the guanidinium group of Arg15 with Asp189 and Gly218 in FXIa. In addition, the Arg15 carbonyl oxygen forms hydrogen bonds with main-chain nitrogen atoms of one of the catalytic triad residues, Ser195, and with Gly193. The other important interaction in FXIa/PN2KPI or trypsin/BPTI is hydrophobic, between PN2KPI-Phe34 and FXIa-Tyr143 and between BPTI-Val34 and trypsin-Tyr151. This intermolecular interaction is further strengthened by an intramolecular interaction in which the side chain of Phe34 packs closely with the side chain of Met17 within PN2KPI, altogether forming a strong hydrophobic patch in FXIa-PN2KPI and trypsin-PN2KPI. PN2KPI-F34A disrupts both inter- and intramolecular hydrophobic interactions, leading to discernable reductions in affinity for both FXIa and trypsin. Despite occupying extreme positions in the autolysis loops, (143YRKLRDKI151 in FXIa and 143NTKSSGTSY151 in trypsin), Tyr143/151 residues still orient themselves in close proximity to Phe34. Thus, loop-1 residues of PN2KPI establish complex ionic interactions that play a major role, which is supplemented by the loop-2 residue, Phe34 (in PN2KPI) or Val34 (in BPTI), which establish hydrophobic interactions with residues in FXIa and trypsin leading to very high-affinity enzyme-inhibitor complexes. Disclosures:No relevant conflicts of interest to declare.

Aryl- and heteroaryl-substituted aminobenzo[ a]quinolizines as dipeptidyl peptidase IV inhibitors

Synthesis and SAR are described for a structurally distinct class of DPP–IV inhibitors based on aminobenzo[ a]quinolizines bearing (hetero-)aromatic substituents in the S1 specificity pocket. The m-(fluoromethyl)-phenyl derivative ( S,S,S)- 2g possesses the best fit in the S1 pocket. However, ( S,S,S)- 2i, bearing a more hydrophilic 5-methyl-pyridin-2-yl residue as substituent for the S1 pocket, displays excellent in vivo activity and superior drug-like properties.

Discovery of carmegliptin: A potent and long-acting dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

Design, synthesis, and SAR are described for a class of DPP-IV inhibitors based on aminobenzo[ a]quinolizines with non-aromatic substituents in the S1 specificity pocket. One representative thereof, carmegliptin ( 8p), was chosen for clinical development. Its X-ray structure in complex with the enzyme and early efficacy data in animal models of type 2 diabetes are also presented.

Chymotrypsin-like peptidases from Tribolium castaneum: A role in molting revealed by RNA interference

Chymotrypsin-like peptidases (CTLPs) of insects are primarily secreted into the gut lumen where they act as digestive enzymes. We studied the gene family encoding CTLPs in the genome of the red flour beetle, Tribolium castaneum. Using an extended search pattern, we identified 14 TcCTLP genes that encode peptidases with S1 specificity pocket residues typically found in chymotrypsin-like enzymes. We further analyzed the expression patterns of seven TcCTLP genes at various developmental stages. While some TcCTLP genes were exclusively expressed in feeding larval and adult stages ( TcCTLP-5A/ B, TcCTLP-6A), others were also detected in non-feeding embryonic ( TcCTLP-5C, TcCTLP-6D) and pupal stages ( TcCTLP-5C, TcCTLP-6C/ D/ E). TcCTLP genes were expressed predominantly in the midgut, where they presumably function in digestion. However, TcCTLP-6C and TcCTLP-5C also showed considerable expression in the carcass. The latter two genes might therefore encode peptidases that act as molting fluid enzymes. To test this hypothesis, we performed western blots using protein extracts from larval exuviae. The extracts reacted with antibodies to TcCTLP-5C and TcCTLP-6E suggesting that the corresponding peptidases are secreted into the molting fluid. Finally, we performed systemic RNAi experiments. While injections of five TcCTLP-dsRNAs into penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects.

Structure and Cleavage Specificity of the Chymotrypsin-Like Serine Protease (3CLSP/nsp4) of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

SummaryBiogenesis and replication of the porcine reproductive and respiratory syndrome virus (PRRSV) include the crucial step of replicative polyprotein processing by self-encoded proteases. Whole genome bioinformatics analysis suggests that nonstructural protein 4 (nsp4) is a 3C-like serine protease (3CLSP), responsible for most of the nonstructural protein processing. The gene encoding this protease was cloned and expressed in Escherichia coli in order to confirm this prediction. The purified protein was crystallized, and the structure was solved at 1.9 Å resolution. In addition, the crystal structure of the Ser118Ala mutant was determined at 2.0 Å resolution. The monomeric enzyme folds into three domains, similar to that of the homologous protease of equine arteritis virus, which, like PRRSV, is a member of the family Arteriviridae in the order of Nidovirales. The active site of the PRRSV 3CLSP is located between domains I and II and harbors a canonical catalytic triad comprising Ser118, His39, and Asp64. The structure also shows an atypical oxyanion hole and a partially collapsed S1 specificity pocket. The proteolytic activity of the purified protein was assessed in vitro. Three sites joining nonstructural protein domains in the PRRSV replicative polyprotein are confirmed to be processed by the enzyme. Two of them, the nsp3/nsp4 and nsp11/nsp12 junctions, are shown to be cleaved in trans, while cis cleavage is demonstrated for the nsp4/nsp5 linker. Thus, we provide structural evidence as well as enzymatic proof of the nsp4 protein being a functional 3CLSP. We also show that the enzyme has a strong preference for glutamic acid at the P1 position of the substrate.

Prostate-specific antigen is a "chymotrypsin-like" serine protease with unique P1 substrate specificity.

Prostate-specific antigen (PSA), a serine protease belonging to the human kallikrein family, is best known as a prostate cancer biomarker. Emerging evidence suggests that PSA may also play a salient role in prostate cancer development and progression. With large amounts of enzymatically active PSA continuously and selectively produced by all stages of prostate cancer, PSA is an attractive target. PSA inhibitors, therefore, may represent a promising class of therapeutics and/or imaging agents. PSA displays chymotrypsin-like specificity, cleaving after hydrophobic residues, in addition to possessing a unique ability to cleave after glutamine in the P1 position. In this study, we investigated the structural motifs of the PSA S1 pocket that give it a distinct architecture and specificity when compared to the S1 pocket of chymotrypsin. Using the previously described PSA substrate Ser-Ser-Lys-Leu-Gln (SSKLQ) as a template, peptide aldehyde based inhibitors containing novel P1 aldehydes were made and tested against both proteases. Glutamine derivative aldehydes were highly specific for PSA while inhibitors with hydrophobic P1 aldehydes were potent inhibitors of both proteases with K(i) values <500 nM. The crystal structure of PSA was used to generate a model that allowed GOLD docking studies to be performed to further understand the critical interactions required for inhibitor binding to the S1 pockets of PSA and chymotrypsin. In conclusion, these results provide experimental and structural evidence that the S1 specificity pocket of PSA is distinctly different from that of chymotrypsin and that the development of highly specific PSA inhibitors is feasible.

Molecular docking identifies the binding of 3-chloropyridine moieties specifically to the S1 pocket of SARS-CoV M pro

The 3C-like main proteinase of the severe acute respiratory syndrome (SARS) coronavirus, SARS-CoV M pro, is widely considered to be a major drug target for the development of anti-SARS treatment. Based on the chemical structure of a lead compound from a previous screening, we have designed and synthesized a number of non-peptidyl inhibitors, some of which have shown significantly improved inhibitory activity against SARS-CoV M pro with IC 50 values of ∼60 nM. In the absence of SARS-CoV M pro crystal structures in complex with these synthetic inhibitors, molecular docking tools have been employed to study possible interactions between these inhibitors and SARS-CoV M pro. The docking results suggest two major modes for the initial binding of these inhibitors to the active site of SARS-CoV M pro. They also establish a structural basis for the ‘core design’ of these inhibitors by showing that the 3-chloropyridine functions common to all of the present inhibitors tend to cluster in the S1 specificity pocket. In addition, intrinsic flexibility in the S4 pocket allows for the accommodation of bulky groups such as benzene rings, suggesting that this structural plasticity can be further exploited for optimizing inhibitor–enzyme interactions that should promote a tighter binding mode. Most importantly, our results provide the structural basis for rational design of wide-spectrum antiviral drugs targeting the chymotrypsin-like cysteine proteinases from coronaviruses and picornaviruses.

Understanding Binding Selectivity toward Trypsin and Factor Xa: the Role of Aromatic Interactions

A congeneric series of four bis-benzamidine inhibitors sharing a dianhydrosugar isosorbide scaffold in common has been studied by crystal structure analysis and enzyme kinetics with respect to their binding to trypsin and factor Xa. Within the series, aromatic interactions are an important determinant for selectivity discrimination among both serine proteases. To study the selectivity-determining features in detail, we used trypsin mutants in which the original binding site is gradually substituted to finally resemble the factor Xa binding pocket. The influence of these mutations has been analyzed on the binding of the closely related inhibitors. We present the crystal structures of the inhibitor complexes obtained by co-crystallizing an "intermediate" trypsin mutant. They could be determined to a resolution of up to 1.2 A, and we measured the inhibitory activity (K(i)) of each ligand against factor Xa, trypsin, and the various mutants. From these data we were able to derive a detailed structure-activity relationship which demonstrates the importance of aromatic interactions in protein-ligand recognition and their role in modulating enzyme selectivity. Pronounced preference is experienced to accommodate the benzamidine anchor with meta topology in the S(1) specificity pocket. One ligand possessing only para topology deviates strongly from the other members of the series and adopts a distinct binding mode addressing the S(1)' site instead of the distal S(3)/S(4) binding pocket.

Alteration of the Factor X Zymogen to Protease Transition Provides Evidence for Allosteric Linkage between the S1 and FVa Binding Sites.

The zymogen to protease transition in the chymotrypsin-like serine protease family follows a well described mechanism in which bond cleavage at a highly conserved site (Arg15-Ile16; chymotrypsin numbering system) results in the unmasking of a new N-terminus that acts as an intramolecular ligand for Asp194. This new salt-bridge drives a conformational change in the so-called “activation domain”, surface loops consisting of the S1 specificity pocket, oxyanion hole, autolysis loop, and sodium biding site. It is well documented in the trypsin system that Ile16-Asp194 internal salt-bridge formation is allosterically linked to the S1 specificity site; that is changes at one site influence the other and vice versa. Blood coagulation factor Xa (FXa) reversibly associates with its cofactor factor Va (FVa) on a membrane surface in the presence of Ca2+ ions with high affinity; an interaction which is not mimicked by the zymogen FX. To determine whether the FX zymogen to protease transition contributes to the expression of a high affinity FVa binding site, we constructed a series of FXa variants which are shifted along this transition pathway. To generate these “zymogen-like” proteins, we made several substitutions at position 16 or 17, with the intent of destabilizing the intramolecular salt bridge to varying degrees. Following a series of preliminary experiments, three mutants were chosen for expression, purification, and activation with RVV-X: I16L, I16G, and V17A. Kinetic studies using peptidyl substrates and active site directed probes revealed that I16L and V17A have an impaired ability to bind these probes (15 to 25-fold increase in the Km or Ki) while the rate of catalysis (kcat) was reduced by 3-fold compared to wild-type FXa (wtFXa; plasma-derived and recombinant). The I16G variant was not inhibited by any of the probes examined and its chromogenic activity was severely impaired (>500 to 1000-fold), precluding calculation of kinetic parameters. These data are consistent with the idea that destabilization of internal salt-bridge formation (Ile16-Asp194) influences binding at the S1 specificity site. In contrast to these results, assembly of I16L and V17A into prothrombinase almost completely restored the Km for peptidyl substrates while the kcat was still 3-fold reduced, indicating that FVa binding can rescue binding at the active site. Surprisingly, even the Km value for I16G was almost completely restored (3-fold increased compared to wtFXa) when assembled in prothrombinase; however a 60-fold reduction in the kcat was found. Consistent with these data, kinetic studies using prothrombin or prethrombin-1 revealed that each of the FXa variants had a normal Km value when assembled in prothrombinase; while the kcat values where reduced to a similar extent as for the chromogenic substrates. Overall our data indicate that direct binding of these FXa variants to FVa rescues binding at S1 site, suggesting allosteric linkage exists between these sites. Thus the FX zymogen to protease transition not only influences the formation of the S1 pocket, but also contributes in a substantial way to the formation of a FVa binding site.

Crystal structure of the β‐chain of human hepatocyte growth factor‐like/macrophage stimulating protein

Hepatocyte growth factor like/macrophage stimulating protein (HGFl/MSP) and hepatocyte growth factor/scatter factor (HGF/SF) define a distinct family of vertebrate-specific growth factors structurally related to the blood proteinase precursor plasminogen and with important roles in development and cancer. Although the two proteins share a similar domain structure and mechanism of activation, there are differences between HGFl/MSP and HGF/SF in terms of the contribution of individual domains to receptor binding. Here we present a crystal structure of the 30 kDa beta-chain of human HGFl/MSP, a serine proteinase homology domain containing the high-affinity binding site for the RON receptor. The structure describes at 1.85 Angstrom resolution the region of the domain corresponding to the receptor binding site recently defined in the HGF/SF beta-chain, namely the central cleft harboring the three residues corresponding to the catalytic ones of active proteinases (numbers in brackets define the sequence position according to the standard chymotrypsinogen numbering system) [Gln522 (c57), Gln568 (c102) and Tyr661 (c195)] and an adjacent loop flanking the S1 specificity pocket and containing residues Asn682 (c217) and Arg683 (c218) previously shown to be essential for binding of HGFl/MSP to the RON receptor. The study confirms the concept that the serine proteinase homology domains of HGFl/MSP and HGF/SF bind their receptors in an 'enzyme-substrate' mode, reflecting the common evolutionary origin of the plasminogen-related growth factors and the proteinases of the clotting and fibrinolytic pathways. However, analysis of the intermolecular interactions in the crystal lattice of beta-chain HGFl/MSP fails to show the same contacts seen in the HGF/SF structures and does not support a conserved mode of dimerization of the serine proteinase homology domains of HGFl/MSP and HGF/SF responsible for receptor activation.