P1 Residue Research Articles

Exploring the active site binding specificity of kallikrein-related peptidase 5 (KLK5) guides the design of new peptide substrates and inhibitors.

Kallikrein-related peptidase 5 (KLK5) is a promising therapeutic target in several skin diseases, including Netherton syndrome, and is emerging as a potential target in various cancers. In this study, we used a sparse matrix library of 125 individually synthesized peptide substrates to characterize the binding specificity of KLK5. The sequences most favored by KLK5 were GRSR, YRSR and GRNR, and we identified sequence-specific interactions involving the peptide N-terminus by analyzing kinetic constants (kcat and KM) and performing molecular dynamics simulations. KLK5 inhibitors were subsequently engineered by substituting substrate sequences into the binding loop (P1, P2 and P4 residues) of sunflower trypsin inhibitor-1 (SFTI-1). These inhibitors were effective against KLK5 but showed limited selectivity, and performing a further substitution at P2' led to the design of a new variant that displayed improved activity against KLK5 (Ki=4.2±0.2 nm), weak activity against KLK7 and 12-fold selectivity over KLK14. Collectively, these findings provide new insight into the design of highly favored binding sequences for KLK5 and reveal several opportunities for modulating inhibitor selectivity over closely related proteases that will be useful for future studies aiming to develop therapeutic molecules targeting KLK5.

Abstract A116: A predictive model to identify immunogenic neoepitopes in patients with melanoma treated with CTLA-4 blockade

Abstract Recent studies have shown that CD8+ T cells that recognize neoantigens presented by tumor cells, but the target specificity of these tumor infiltrating T cells are largely unknown. Developing a predictive model that identifies immunogenic neoepitopes presented by major histocompatibility complex (MHC) class I molecules is critical to identify potential vaccine targets and to improve prediction of response to immune checkpoint inhibitors. We hypothesize that the immunogenic heterogeneity of tumors may explain, in part, differences in clinical response that individual patients derive to checkpoint blockade therapy. We have recently shown that the relative hydrophobicity of T cell receptor (TCR) contact residues is a hallmark of immunogenic MHC class I epitopes, and that this may represent a biochemical basis by which T cells discriminate immunogenic epitopes within the background of self-peptides. Here, we extend this analysis using candidate MHC class I tumor neopeptides from patients with melanoma. Somatic missense mutations were identified from recently published data on whole-exome sequencing of tumors from 11 patients who derived a long-term benefit and 14 patients who derived a minimal or no benefit from ipilimumab or tremelimumab. Mutational burden ranged from 1 to 1,304 mutations per tumor. More than 3,200 potential MHC class I neopeptides were identified based on a predicted binding algorithm (binding affinity, ≤500 nM). Our preliminary results indicate significant differences in hydrophobicity at the anchor residues P2 and P9 (P < 0.001 for both), reflecting MHC binding differences. In addition, there is an increase in hydrophobicity at the TCR contact residues P4 and P7 (P < 0.01 for both) in long-term responders. The overall frequency of mutations in neoepitopes is significantly different at the TCR contact residues P4 and P7 between long-term responders and minimal or no responders (P < 0.01), suggesting genetic immunoediting of these tumors. Based on these results, we have developed a probabilistic Bayesian-based model to predict immunogenic neoepitopes. The model is being applied on the overall pool of neopeptides from each individual patient with melanoma. The sets of predicted immunogenic neoepitopes may serve as potential therapeutic vaccine candidates for patients with melanoma. Citation Format: Diego Chowell, Sri Krishna, Alexandra Snyder, Makarov Vladimir, Timothy A. Chan, Karen S. Anderson. A predictive model to identify immunogenic neoepitopes in patients with melanoma treated with CTLA-4 blockade. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A116.

Insights into the serine protease mechanism based on structural observations of the conversion of a peptidyl serine protease inhibitor to a substrate

BackgroundSerine proteases are one of the most studied group of enzymes. Despite the extensive mechanistic studies, some crucial details remain controversial, for example, how the cleaved product is released in the catalysis reaction. A cyclic peptidyl inhibitor (CSWRGLENHRMC, upain-1) of a serine protease, urokinase-type plasminogen activator (uPA), was found to become a slow substrate and cleaved slowly upon the replacement of single residue (W3A). MethodsBy taking advantage of the unique property of this peptide, we report the high-resolution structures of uPA in complex with upain-1-W3A peptide at four different pH values by X-ray crystallography. ResultsIn the structures obtained at low pH (pH4.6 and 5.5), the cyclic peptide upain-1-W3A was found to be intact and remained in the active site of uPA. At 7.4, the scissile bond of the peptide was found cleaved, showing that the peptide became a uPA substrate. At pH9.0, the C-terminal part of the substrate was no longer visible, and only the P1 residue occupying the S1 pocket was identified. ConclusionsThe analysis of these structures provides explanations why the upain-1-W3A is a slow substrate. In addition, we clearly identified the cleaved fragments of the peptide at both sides of the scissile bond in the active site of the enzyme, showing a slow release of the cleaved peptide. General significanceThis work indicates that the quick release of the cleaved P′ fragment after the first step of hydrolysis may not always be needed for the second hydrolysis.

Active site-directed plasmin inhibitors: Extension on the P2 residue

Based on the structure of YO-2 [N-(trans-4-aminomethylcyclohexanecarbonyl)-l-Tyr(O-picolyl)-NH-octyl], active site-directed plasmin (Plm) inhibitors were explored. The picolyl moiety in the Tyr(O-picolyl) residue (namely, the P2 residue) was replaced with smaller or larger groups, such as hydrogen, tert-butyl, benzyl, (2-naphthyl)methyl, and (quinolin-2-yl)methyl. Those efforts produced compound 17 {N-(trans-4-aminomethylcyclohexanecarbonyl)-l-Tyr[O-(quinolin-2-yl)methyl]-NH-octyl} [IC50=0.22 and 77μM for Plm and urokinase (UK), respectively], which showed not only 2.4-fold greater Plm inhibition than YO-2, but also an improvement in selectivity (Plm/UK) by 35-fold. The docking experiments of the Plm-17 complexes disclosed that the amino group of the tranexamyl moiety interacted with the side-chain of Asp753 which formed S1 site.

Identification of CD4+ T-cell epitopes on iron-regulated surface determinant B of Staphylococcus aureus

Iron-regulated surface determinant B (IsdB) of Staphylococcus aureus (S. aureus) is a highly conserved surface protein that can induce protective CD4+ T-cell immune response. A pivotal role of CD4+ T-cells in effective immunity against S. aureus infection has been proved, but CD4+ T-cell epitopes on the S. aureus IsdB have not been well identified. In this study, MHC binding assay was firstly used to predict CD4+ T-cell epitopes on S. aureus IsdB protein, and six peptides were synthesized to validate the probable epitopes. Two novel IsdB CD4+ T-cell epitopes, P1 (residues 159–178) and P4 (residues 287–306), were for the first time identified using CD4+ T-cells obtained from IsdB-immunized C57BL/6 (H-2b) and BALB/c (H-2d) mice spleen based on cell proliferation and cytokines response. The results showed that P1 and P4 emulsified in Freund's adjuvant (FA) induced much higher cell proliferation compared with PBS emulsified in FA. CD4+ T-cells stimulated with peptides P1 and P4 secreted significantly higher levels of IFN-γ and IL-17A. However, the level of the cytokine IL-4 almost remained unchanged, suggesting that P1 and P4 preferentially elicited polarized Th1-type responses. In addition, BALB/c mice just respond to P4 not P1, while C57BL/6 mice respond to P1 not P4, implying that epitope P1 and P4 were determined as H-2b and H-2d restricted epitope, respectively. Taken together, our data may provide an explanation of the IsdB-induced protection against S. aureus and highlight the possibility of developing the epitope-based vaccine against the S. aureus.

Exopeptidases and gingipains in Porphyromonas gingivalis as prerequisites for its amino acid metabolism.

SummaryPorphyromonas gingivalis, an asaccharolytic bacterium, utilizes amino acids as energy and carbon sources. Since amino acids are incorporated into the bacterial cells mainly as di- and tri-peptides, exopeptidases including dipeptidyl-peptidase (DPP) and tripeptidyl-peptidase are considered to be prerequisite components for their metabolism. We recently discovered DPP11, DPP5, and acylpeptidyl oligopeptidase in addition to previously reported DPP4, DPP7, and prolyl tripeptidyl peptidase A. DPP11 is a novel enzyme specific for acidic P1 residues (Asp and Glu) and distributed ubiquitously in eubacteria, while DPP5 is preferential for the hydrophobic P1 residue and the first entity identified in prokaryotes. Recently, acylpeptidyl oligopeptidase with a preference for hydrophobic P1 residues was found to release N-terminally blocked di- and tri-peptides. Furthermore, we also demonstrated that gingipains R and K contribute to P1-basic dipeptide production. These observations implicate that most, if not all, combinations of di- and tri-peptides are produced from extracellular oligopeptides even with an N-terminal modification. Here, we review P. gingivalis exopeptidases mainly in regard to their enzymatic characteristics. These exopeptidases with various substrate specificities benefit P. gingivalis for obtaining energy and carbon sources from the nutritionally limited subgingival environment.

Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses

Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6′ in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1′ (with a ~32–93% preference for leucine depending on the MMP), and basic and small residues in P2′ and P3′, respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1′ leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1′-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with leucine locked in S1′. Similar negative cooperativity between P3 proline and the novel preference for asparagine in P1 cements our conclusion that non-prime side flexibility greatly impacts MMP binding affinity and cleavage efficiency. Thus, unexpected sequence cooperativity consequences were revealed by PICS that uniquely encompasses both the non-prime and prime sides flanking the proteomic-pinpointed scissile bond.

Structural and computational analysis of peptide recognition mechanism of class-C type penicillin binding protein, alkaline D-peptidase from Bacillus cereus DF4-B.

Alkaline D-peptidase from Bacillus cereus DF4-B, called ADP, is a D-stereospecific endopeptidase reacting with oligopeptides containing D-phenylalanine (D-Phe) at N-terminal penultimate residue. ADP has attracted increasing attention because it is useful as a catalyst for synthesis of D-Phe oligopeptides or, with the help of substrate mimetics, L-amino acid peptides and proteins. Structure and functional analysis of ADP is expected to elucidate molecular mechanism of ADP. In this study, the crystal structure of ADP (apo) form was determined at 2.1 Å resolution. The fold of ADP is similar to that of the class C penicillin-binding proteins of type-AmpH. Docking simulations and fragment molecular orbital analyses of two peptides, (D-Phe)4 and (D-Phe)2-(L-Phe)2, with the putative substrate binding sites of ADP indicated that the P1 residue of the peptide interacts with hydrophobic residues at the S1 site of ADP. Furthermore, molecular dynamics simulation of ADP for 50 nsec suggested that the ADP forms large cavity at the active site. Formation of the cavity suggested that the ADP has open state in the solution. For the ADP, having the open state is convenient to bind the peptides having bulky side chain, such as (D-Phe)4. Taken together, we predicted peptide recognition mechanism of ADP.

Phosphoramidate-based peptidomimetic inhibitors of membrane type-1 matrix metalloproteinase

Membrane-type I matrix metalloproteinases (MT1-MMP) is an enzyme critical to the remodeling and homeostasis of extracellular matrix, and when over expressed it contributes to metastasis and cancer cell progression. Because of its role and implication as a biomarker that is upregulated in various cancers, MT1-MMP has become an attractive target for drug discovery. A small pilot library of peptidomimetics containing a phosphoramidate core as a zinc-binding group was synthesized and tested for inhibitory potency against MT1-MMP. From this library, a novel two residue peptidomimetic scaffold was identified that confers potency against MT1-MMP at submicromolar concentrations. The results of this study confirm that for this scaffold, valine is favored as a P1 residue and leucine in the P1′ position. Furthermore, steric tolerance was observed for the N-terminus, thus implicating that a second-generation library could be constructed to extend the scaffold to P2 without concomitant loss of affinity within the MT1-MMP catalytic domain.

Independent amino acid residues in the S2 pocket of falcipain-3 determine its specificity for P2 residues in substrates

Falcipain-3 (FP3) is an essential and drug target cysteine protease of the most lethal human malaria parasite Plasmodium falciparum. FP3 and its majority of homologs in malaria parasites prefer Leu at the P2 position in substrates and inhibitors, whereas its major host homolog cathepsin L prefers Phe. However, FP3 is much less active on peptide substrates and has negligible activity against a P2 Arg-containing substrate (Z-RR-AMC) compared to its paralog falcipain-2A (FP2A). To identify the specificity determinants, the S2/3 pocket residues of FP3 were substituted with the corresponding residues in FP2 or cathepsin L, and the wild type and mutant proteases were assessed for hydrolysis of peptide and protein substrates. Our results indicate that the S2 pocket residues I94 and P181 of FP3 are chiefly responsible for its P2 Leu preference and negligible activity for Z-RR-AMC, respectively. E243 in FP3 and the corresponding residue D234 in FP2 have a key role in Z-RR-AMC hydrolysing activity, possibly through stabilization of side chain interactions, as their substitution with Ala abolished the activity. Several FP3 mutants, which retained P2 Leu preference and showed similar or more activity than wild type FP3 on peptide substrates, degraded haemoglobin less efficiently than wild type FP3, suggesting that multiple residues contribute to haemoglobinase activity. Furthermore, P181 and E243 appear to contribute to the optimum activity of FP3 in the food vacuole milieu (≈pH 5.5). The identification of residues determining specificity of FP3 could aid in developing specific inhibitors of FP3 and its homologs in malaria parasites.

Identities of P2 and P3 Residues of H-2Kb-Bound Peptides Determine Mouse Ly49C Recognition.

Ly49 receptors can be peptide selective in their recognition of MHC-I-peptide complexes, affording them a level of discrimination beyond detecting the presence or absence of specific MHC-I allele products. Despite this ability, little is understood regarding the properties that enable some peptides, when bound to MHC-I molecules, to support Ly49 recognition, but not others. Using RMA-S target cells expressing MHC-I molecules loaded with individual peptides and effector cells expressing the ectodomain of the inhibitory Ly49C receptor, we found that two adjacent amino acid residues, P2 and P3, both buried in the peptide binding groove of H-2Kb, determine mouse Ly49C specificity. If both are aliphatic residues, this is supportive. Whereas, small amino acids at P2 and aromatic amino acids at the P3 auxiliary anchor residue are detrimental to Ly49C recognition. These results resemble those with a rat Ly49 where the identity of a peptide anchor residue determines recognition, suggesting that dependence on specific peptide residues buried in the MHC-I peptide-binding groove may be fundamental to Ly49 peptide selectivity and recognition.

Smac-Derived Aza-Peptide As an Aminopeptidase-Resistant XIAP BIR3 Antagonist

The peptidic nature of anti-IAPs N-terminus Smac-derived peptides precludes their utilization as potential therapeutic anticancer agents. Recent advances in the development of novel Smacderived peptidomimetics and non-peptidic molecules with improved anti-IAPs activity and resistance to proteolytic cleavage have been reported and led to a number of candidates that are currently in clinical trials including LCL-161, SM-406/AT-406, GDC-0512/GDC-0917, and birinapant. As an attempt to improve the proteolytic stability of Smac peptides, we developed the Aza-peptide AzaAla- Val-Pro-Phe-Tyr-NH2 (2). Unlike unmodified peptide Ala-Val-Pro-Phe-Tyr-NH2 (1), analogue (2) exhibited resistance towards proteolytic cleavage by two aminopeptidases; LAP and DPP-IV, while retaining its IAP inhibitory activity. This was due to the altered planar geometry of the P1 residue side chain. Our findings showed that using aza-isosteres of bioactive peptide sequences imbue the residue with imperviousness to proteolysis; underscoring a potential approach for developing a new generation of Smac-derived Aza-peptidomimetics.

Comparison of mammalian and bacterial expression library screening to detect recombinant alpha-1 proteinase inhibitor variants with enhanced thrombin inhibitory capacity☆

Serpins are a widely distributed family of serine proteases. A key determinant of their specificity is the reactive centre loop (RCL), a surface motif of ∼20 amino acids in length. Expression libraries of variant serpins could be rapidly probed with proteases to develop novel inhibitors if optimal systems were available. The serpin variant alpha-1 proteinase inhibitor M358R (API M358R) inhibits the coagulation protease thrombin, but at sub-maximal rates compared to other serpins. Here we compared two approaches to isolate functional API variants from serpin expression libraries, using the same small library of API randomized at residue 358 (M358X): flow cytometry of transfected HEK 293 cells expressing membrane-displayed API; and a thrombin capture assay (TCA) performed on pools of bacterial lysates expressing soluble API. No enrichment for specific P1 residues was observed when the RCL codons of the 1% of sorted transfected 293 cells with the highest fluorescent thrombin-binding signals were subcloned and sequenced. In contrast, screening of 16 pools of bacterial API-expressing transformants led to the facile identification of API M358R and M358K as functional variants. Kinetic characterization showed that API M358R inhibited thrombin 17-fold more rapidly than API M358K. Reducing the incubation time with immobilized thrombin improved the sensitivity of TCA to detect supra-active API M358R variants and was used to screen a hypervariable library of API variants expressing 16 different amino acids at residues 352–357. The most active variant isolated, with TLSATP substituted for FLEAI, inhibited thrombin 2.9-fold more rapidly than API M358R. Our results indicate that flow cytometric approaches used in protein engineering of antibodies are not appropriate for serpins, and highlight the utility of the optimized TCA for serpin protein engineering.

Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

Elastase-like enzymes are involved in important diseases such as acute pancreatitis, chronic inflammatory lung diseases, and cancer. Structural insights into their interaction with specific inhibitors will contribute to the development of novel anti-elastase compounds that resist rapid oxidation and proteolysis. Proteinaceous Kunitz-type inhibitors homologous to the bovine pancreatic trypsin inhibitor (BPTI) provide a suitable scaffold, but the structural aspects of their interaction with elastase-like enzymes have not been elucidated. Here, we increased the selectivity of ShPI-1, a versatile serine protease inhibitor from the sea anemone Stichodactyla helianthus with high biomedical and biotechnological potential, toward elastase-like enzymes by substitution of the P1 residue (Lys(13)) with leucine. The variant (rShPI-1/K13L) exhibits a novel anti-porcine pancreatic elastase (PPE) activity together with a significantly improved inhibition of human neuthrophil elastase and chymotrypsin. The crystal structure of the PPE·rShPI-1/K13L complex determined at 2.0 Å resolution provided the first details of the canonical interaction between a BPTI-Kunitz-type domain and elastase-like enzymes. In addition to the essential impact of the variant P1 residue for complex stability, the interface is improved by increased contributions of the primary and secondary binding loop as compared with similar trypsin and chymotrypsin complexes. A comparison of the interaction network with elastase complexes of canonical inhibitors from the chelonian in family supports a key role of the P3 site in ShPI-1 in directing its selectivity against pancreatic and neutrophil elastases. Our results provide the structural basis for site-specific mutagenesis to further improve the binding affinity and/or direct the selectivity of BPTI-Kunitz-type inhibitors toward elastase-like enzymes.

Synthesis and evaluation of constrained phosphoramidate inhibitors of prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA) is a cell-surface enzyme-biomarker that is actively pursued for targeted delivery of imaging and therapeutic agents for prostate cancer. Our lab has developed PSMA inhibitors based on a phosphoramidate scaffold, which has shown both high selectivity for PSMA-positive tumors and rapid clearance in vivo when radiolabeled with (18)F. However, this scaffold exhibits hydrolytic instability under low pH and high temperature conditions, barring the use of other imaging or therapeutic radionuclides such as (68)Ga or (177)Lu. Previous studies in our lab have shown a trend in increasing acid stability as the distance between the phosphoramidate core and the α-carboxylate of the P1 residue is increased. Therefore, a new generation of phosphoramidate inhibitors was developed based on trans-4-hydroxyproline as the P1 residue to restrict the interaction of the α-carboxylate to the phosphoramidate core. These hydroxyproline inhibitors demonstrated comparable IC50 values to earlier generations as well as enhanced thermal and acid stability.

Structural basis for the inhibition of M1 family aminopeptidases by the natural product actinonin: Crystal structure in complex with E. coli aminopeptidase N.

Actinonin is a pseudotripeptide that displays a high affinity towards metalloproteases including peptide deformylases (PDFs) and M1 family aminopeptidases. PDF and M1 family aminopeptidases belong to thermolysin-metzincin superfamily. One of the major differences in terms of substrate binding pockets between these families is presence (in M1 aminopeptidases) or absence (in PDFs) of an S1 substrate pocket. The binding mode of actinonin to PDFs has been established previously; however, it is not clear how the actinonin, without a P1 residue, would bind to the M1 aminopeptidases. Here we describe the crystal structure of Escherichia coli aminopeptidase N (ePepN), a model protein of the M1 family aminopeptidases in complex with actinonin. For comparison we have also determined the structure of ePepN in complex with a well-known tetrapeptide inhibitor, amastatin. From the comparison of the actinonin and amastatin ePepN complexes, it is clear that the P1 residue is not critical as long as strong metal chelating head groups, like hydroxamic acid or α-hydroxy ketone, are present. Results from this study will be useful for the design of selective and efficient hydroxamate inhibitors against M1 family aminopeptidases.

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.

PtPLC, a pacifastin-related inhibitor involved in antibacterial defense and prophenoloxidase cascade of the swimming crab Portunus trituberculatus

Pacifastin-related inhibitor is a new family of serine protease inhibitors that regulate the proteolytic cascade in multiple biological processes. Contrary to the knowledge on the structure and inhibitory mechanism of pacifastin-like members in locust, very little is known about their functions. Here, we report the inhibitory activities in relation to the structural characteristics of pacifastin light chain (PtPLC) gene identified from the swimming crab Portunus trituberculatus. The mature PtPLC and five PLD-related domains with critical residues were expressed in Escherichia coli, and assayed for their activities. The recombinant PtPLC (rPtPLC) displayed inhibitory activities against trypsin and chymotrypsin in a dose dependent manner, with a preference for trypsin. Except for rPtPLC-D4, the other four rPtPLC-related domains could inhibit at least one of serine proteases. The enzyme specificity of PtPLC domains generally corresponded to the nature of the P1 residue at the reactive site. rPtPLC was able to inhibit the growth of Gram-negative bacteria Vibrio alginolyticus and Pseudomonas aeruginosa, but not the Gram-positive bacterium and fungus tested. Further phenoloxidase (PO) assay showed the rPtPLC could depress the crab proPO system activation in vitro, and lead to 72.8% inhibition of PO activity at the concentration of 9.11 μM. It also suppressed proPO activation induced by rPtcSP and rPtSPH1. As the first functional study of the recombinant PLC protein in crustaceans, the present results together indicate that PtPLC functions in the crab immune response possibly via inhibiting bacterial growth and regulating the proPO system.

Isolation and characterization of a protease inhibitor from Acacia karroo with a common combining loop and overlapping binding sites for chymotrypsin and trypsin

By using affinity and reversed-phase HPLC (RP-HPLC) chromatographies two chymotrypsin–trypsin inhibitors were isolated from seeds of Acacia karroo, a legume of the subfamily Mimosoideae. The primary structure of one of these inhibitors, named AkCI/1, was determined. The inhibitor consists of two polypeptide chains, 139 and 44 residues respectively, which are linked by a single disulfide bridge. The amino acid sequence of AkCI/1 is homologous to and showed more than 60% sequence similarity with other protease inhibitors isolated earlier from the group of Mimosoideae. AkCI/1 inhibits both chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4) in a 1:1M ratio with Ki values of 2.8×10−12M and 1.87×10−12M, respectively. The P1–P1′ residues for trypsin were identified as Arg68-Ile69 by selective hydrolysis of the inhibitor at this site, with bovine trypsin and human trypsin IV. The cleavage did not affect the inhibition of trypsin, but fully abolished the chymotrypsin inhibitory activity of AkCI/1. This finding together with our studies on competition of the two enzymes for the same combining loop suggests that the same loop has to contain the binding sites for both proteases. The most likely P1 residue of AkCI/1 for chymotrypsin is Tyr67.