Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P₁ (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P′₂ favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P₁ and P′₂ substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin·APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.     

A Protease Inhibitor of the Serpin Family Is a Major Protein in Carp Perimeningeal Fluid: I. Protein Purification and Characterization

Abstract: Two isoforms of a protease inhibitor of the serpin family (p62) have been purified from bighead carp perimeningeal fluid. Both isoforms migrate with an apparent molecular mass of 62 kDa on reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gels. Both proteins inhibited the activities of bovine trypsin, bovine chymotrypsin, and porcine pancreatic elastase. They also formed complexes with these proteases that were resistant to sodium dodecyl sulfate treatment. p62 exists in the extracts of all tissues examined, including brain, head kidney, kidney, liver, muscle, ovary, pituitary, and spleen. It is also present in serum, ovarian fluid, and milt as well as perimeningeal fluid. The protease inhibitor is a glycoprotein, and its carbohydrate moiety could be removed by endoglycosidase F. Because p62 resembles mammalian α₁-antitrypsin in many aspects, it is likely a fish equivalent of α₁-antitrypsin.     

Blastocystis Legumain Is Localized on the Cell Surface, and Specific Inhibition of Its Activity Implicates a Pro-survival Role for the Enzyme

Programmed cell death (PCD) is crucial for cellular growth and development in multicellular organisms. Although distinct PCD features have been described for unicellular eukaryotes, homology searches have failed to reveal clear PCD-related orthologues among these organisms. Our previous studies revealed that a surface-reactive monoclonal antibody (mAb) 1D5 could induce multiple PCD pathways in the protozoan Blastocystis. In this study, we identified, by two-dimensional gel electrophoresis and mass spectrometry, the target of mAb 1D5 as a surface-localized legumain, an asparagine endopeptidase that is usually found in lysosomal/acidic compartments of other organisms. Recombinant Blastocystis legumain displayed biphasic pH optima in substrate assays, with peaks at pH 4 and 7.5. Activity of Blastocystis legumain was greatly inhibited by the legumain-specific inhibitor carbobenzyloxy-Ala-Ala-AAsn-epoxycarboxylate ethyl ester (APE-RR) (where AAsn is aza-asparagine) and moderately inhibited by mAb 1D5, cystatin, and caspase-1 inhibitor. Interestingly, inhibition of legumain activity induced PCD in Blastocystis, observed by increased externalization of phosphatidylserine residues and in situ DNA fragmentation. In contrast to plants, in which legumains have been shown to play a pro-death role, legumain appears to display a pro-survival role in Blastocystis.     

The role of factor XIa (FXIa) catalytic domain exosite residues in substrate catalysis and inhibition by the Kunitz protease inhibitor domain of protease nexin 2

To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu(98), Tyr(143), Ile(151), Arg(3704), Lys(192), and Tyr(5901)) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal K(m) values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of k(cat) for S-2366 hydrolysis. All six Ala mutants displayed deficient k(cat) values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of K(i) except for K192A, and Y5901A, which displayed increased values of K(i). The integrity of the S1 binding site residue, Asp(189), utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr(143), Ile(151), Arg(3704), and Tyr(5901)) are important for S-2366 hydrolysis; Glu(98) and Lys(192) are essential for FIX but not S-2366 hydrolysis; and Lys(192) and Tyr(5901) are required for both inhibitor and macromolecular substrate interactions.     

The Novel Membrane Protein TMEM59 Modulates Complex Glycosylation,Cell Surface Expression,and Secretion of the Amyloid Precursor Protein

Ectodomain shedding of the amyloid precursor protein (APP) by the two proteases α- and β-secretase is a key regulatory event in the generation of the Alzheimer disease amyloid β peptide (Aβ). At present, little is known about the cellular mechanisms that control APP shedding and Aβ generation. Here, we identified a novel protein, transmembrane protein 59 (TMEM59), as a new modulator of APP shedding. TMEM59 was found to be a ubiquitously expressed, Golgi-localized protein. TMEM59 transfection inhibited complex N- and O-glycosylation of APP in cultured cells. Additionally, TMEM59 induced APP retention in the Golgi and inhibited Aβ generation as well as APP cleavage by α- and β-secretase cleavage, which occur at the plasma membrane and in the endosomes, respectively. Moreover, TMEM59 inhibited the complex N-glycosylation of the prion protein, suggesting a more general modulation of Golgi glycosylation reactions. Importantly, TMEM59 did not affect the secretion of soluble proteins or the α-secretase like shedding of tumor necrosis factor α, demonstrating that TMEM59 did not disturb the general Golgi function. The phenotype of TMEM59 transfection on APP glycosylation and shedding was similar to the one observed in cells lacking conserved oligomeric Golgi (COG) proteins COG1 and COG2. Both proteins are required for normal localization and activity of Golgi glycosylation enzymes. In summary, this study shows that TMEM59 expression modulates complex N- and O-glycosylation and suggests that TMEM59 affects APP shedding by reducing access of APP to the cellular compartments, where it is normally cleaved by α- and β-secretase.     

The Allosteric Mechanism of Activation of Antithrombin as an Inhibitor of Factor IXa and Factor Xa: HEPARIN-INDEPENDENT FULL ACTIVATION THROUGH MUTATIONS ADJACENT TO HELIX D*

Allosteric conformational changes in antithrombin induced by binding a specific heparin pentasaccharide result in very large increases in the rates of inhibition of factors IXa and Xa but not of thrombin. These are accompanied by CD, fluorescence, and NMR spectroscopic changes. X-ray structures show that heparin binding results in extension of helix D in the region 131–136 with coincident, and possibly coupled, expulsion of the hinge of the reactive center loop. To examine the importance of helix D extension, we have introduced strong helix-promoting mutations in the 131–136 region of antithrombin (YRKAQK to LEEAAE). The resulting variant has endogenous fluorescence indistinguishable from WT antithrombin yet, in the absence of heparin, shows massive enhancements in rates of inhibition of factors IXa and Xa (114- and 110-fold, respectively), but not of thrombin, together with changes in near- and far-UV CD and ¹H NMR spectra. Heparin binding gives only ∼3–4-fold further rate enhancement but increases tryptophan fluorescence by ∼23% without major additional CD or NMR changes. Variants with subsets of these mutations show intermediate activation in the absence of heparin, again with basal fluorescence similar to WT and large increases upon heparin binding. These findings suggest that in WT antithrombin there are two major complementary sources of conformational activation of antithrombin, probably involving altered contacts of side chains of Tyr-131 and Ala-134 with core hydrophobic residues, whereas the reactive center loop hinge expulsion plays only a minor additional role.     

Interactions Outside the Proteinase-binding Loop Contribute Significantly to the Inhibition of Activated Coagulation Factor XII by Its Canonical Inhibitor from Corn

Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (K_i = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a K_i of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20–45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (K_i = 11.7 ± 1.2 μm) and activated factor XI (K_i = 94 ± 11 μm). Full-length CHFI inhibited trypsin with a K_i of 1.3 ± 0.2 nm and activated factor XI with a K_i of 5.4 ± 0.2 μm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.     

In Vivo Neuronal Synthesis and Axonal Transport of Kunitz Protease Inhibitor (KPI)-Containing Forms of the Amyloid Precursor Protein

Abstract: We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.     

Heparin is a major activator of the anticoagulant serpin, protein Z-dependent protease inhibitor

Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa and XIa. Here we provide evidence that, in addition to the established cofactors, protein Z, lipid, and calcium, heparin is an important cofactor of ZPI anticoagulant function. Heparin produced 20-100-fold accelerations of ZPI reactions with factor Xa and factor XIa to yield second order rate constants approaching the physiologically significant diffusion limit (k(a) = 10(6) to 10(7) M(-1) s(-1)). The dependence of heparin accelerating effects on heparin concentration was bell-shaped for ZPI reactions with both factors Xa and XIa, consistent with a template-bridging mechanism of heparin rate enhancement. Maximal accelerations of ZPI-factor Xa reactions required calcium, which augmented the heparin acceleration by relieving Gla domain inhibition as previously shown for heparin bridging of the antithrombin-factor Xa reaction. Heparin acceleration of both ZPI-protease reactions was optimal at heparin concentrations and heparin chain lengths comparable with those that produce physiologically significant rate enhancements of other serpin-protease reactions. Protein Z binding to ZPI minimally affected heparin rate enhancements, indicating that heparin binds to a distinct site on ZPI and activates ZPI in its physiologically relevant complex with protein Z. Taken together, these results suggest that whereas protein Z, lipid, and calcium cofactors promote ZPI inhibition of membrane-associated factor Xa, heparin activates ZPI to inhibit free factor Xa as well as factor XIa and therefore may play a physiologically and pharmacologically important role in ZPI anticoagulant function.     

The N-terminal Arg Residue Is Essential for Autocatalytic Activation of a Lipopolysaccharide-responsive Protease Zymogen

Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules.     

Functional Analysis of the Cucumisin Propeptide as a Potent Inhibitor of Its Mature Enzyme

Cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (Cucumis melo L.). It is synthesized as a preproprotein consisting of a signal peptide, NH₂-terminal propeptide, and 67-kDa protease domain. We investigated the role of this propeptide (88 residues) in the cucumisin precursor. Complementary DNAs encoding the propeptides of cucumisin, two other plant subtilases (Arabidopsis ARA12 and rice RSP1), and bacterial subtilisin E were expressed in Escherichia coli independently of their mature enzymes. The cucumisin propeptide strongly inhibited cucumisin in a competitive manner with a K_i value of 6.2 ± 0.55 nm. Interestingly, cucumisin was also strongly inhibited by ARA12 and RSP1 propeptides but not by the subtilisin E propeptide. In contrast, the propeptides of cucumisin, ARA12, and RSP1 did not inhibit subtilisin. Deletion analysis clearly showed that two hydrophobic regions, Asn³²–Met³⁸ and Gly⁹⁷–Leu¹⁰³, in the cucumisin propeptide were important for its inhibitory activity. Site-directed mutagenesis also confirmed the role of a Val³⁶-centerd hydrophobic cluster within the Asn³²–Met³⁸ region in cucumisin inhibition. Circular dichroism spectroscopy revealed that the cucumisin propeptide had a secondary structure without a cognate protease domain and that the thermal unfolding of the propeptide at 90 °C was only partial and reversible. A tripeptide, Ile³⁵-Val³⁶-Tyr³⁷, in the Asn³²–Met³⁸ region was thought to contribute toward the formation of a proper secondary structure necessary for cucumisin inhibition. This is the first report on the function and structural information of the propeptide of a plant serine protease.     

The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O_δ2–C_γ bond appears to be a double bond, with O_δ2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O_δ1 atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF_obs − DF_calc density above 2.5 σ next to O_δ1. As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK_a of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK_a histidine theory.     

Specific Cleavage of the Nuclear Pore Complex Protein Nup62 by a Viral Protease

Previous work has shown that several nucleoporins, including Nup62 are degraded in cells infected with human rhinovirus (HRV) and poliovirus (PV) and that this contributes to the disruption of certain nuclear transport pathways. In this study, the mechanisms underlying proteolysis of Nup62 have been investigated. Analysis of Nup62 in lysates from HRV-infected cells revealed that Nup62 was cleaved at multiple sites during viral infection. The addition of purified HRV2 2A protease (2A^pro) to uninfected HeLa whole cell lysates resulted in the cleavage of Nup62, suggesting that 2A^pro is a major contributor to Nup62 processing. The ability of purified 2A^pro to cleave bacterially expressed and purified Nup62 demonstrated that 2A^pro directly cleaves Nup62 in vitro. Site-directed mutagenesis of putative cleavage sites in Nup62 identified six different positions that are cleaved by 2A^pro in vitro. This analysis revealed that 2A^pro cleavage sites were located between amino acids 103 and 298 in Nup62 and suggested that the N-terminal FG-rich region of Nup62 was released from the nuclear pore complex in infected cells. Analysis of HRV- and PV-infected cells using domain-specific antibodies confirmed that this was indeed the case. These results are consistent with a model whereby PV and HRV disrupt nucleo-cytoplasmic trafficking by selectively removing FG repeat domains from a subset of nuclear pore complex proteins.     

Isolation,cDNA Cloning,and Structure-based Functional Characterization of Oryctin,a Hemolymph Protein from the Coconut Rhinoceros Beetle,Oryctes rhinoceros,as a Novel Serine Protease Inhibitor

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant ¹³C,¹⁵N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with K_i values of 3.9 × 10⁻¹⁰ m, 6.2 × 10⁻¹⁰ m, 1.4 × 10⁻⁹ m, and 1.2 × 10⁻⁸ m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections.     

Foamy Virus Envelope Protein Is a Substrate for Signal Peptide Peptidase-like 3 (SPPL3)

Signal peptide peptidase (SPP), its homologs, the SPP-like proteases SPPL2a/b/c and SPPL3, as well as presenilin, the catalytic subunit of the γ-secretase complex, are intramembrane-cleaving aspartyl proteases of the GxGD type. In this study, we identified the 18-kDa leader peptide (LP18) of the foamy virus envelope protein (FVenv) as a new substrate for intramembrane proteolysis by human SPPL3 and SPPL2a/b. In contrast to SPPL2a/b and γ-secretase, which require substrates with an ectodomain shorter than 60 amino acids for efficient intramembrane proteolysis, SPPL3 cleaves mutant FVenv lacking the proprotein convertase cleavage site necessary for the prior shedding. Moreover, the cleavage product of FVenv generated by SPPL3 serves as a new substrate for consecutive intramembrane cleavage by SPPL2a/b. Thus, human SPPL3 is the first GxGD-type aspartyl protease shown to be capable of acting like a sheddase, similar to members of the rhomboid family, which belong to the class of intramembrane-cleaving serine proteases.     

Molecular Mechanism by Which a Potent Hepatitis C Virus NS3-NS4A Protease Inhibitor Overcomes Emergence of Resistance

Although optimizing the resistance profile of an inhibitor can be challenging, it is potentially important for improving the long term effectiveness of antiviral therapy. This work describes our rational approach toward the identification of a macrocyclic acylsulfonamide that is a potent inhibitor of the NS3-NS4A proteases of all hepatitis C virus genotypes and of a panel of genotype 1-resistant variants. The enhanced potency of this compound versus variants D168V and R155K facilitated x-ray determination of the inhibitor-variant complexes. In turn, these structural studies revealed a complex molecular basis of resistance and rationalized how such compounds are able to circumvent these mechanisms.     

Molecular Design of a Highly Selective and Strong Protein Inhibitor against Matrix Metalloproteinase-2 (MMP-2)

Synthetic inhibitors of matrix metalloproteinases (MMPs), designed previously, as well as tissue inhibitors of metalloproteinases (TIMPs) lack enzyme selectivity, which has been a major obstacle for developing inhibitors into safe and effective MMP-targeted drugs. Here we designed a fusion protein named APP-IP-TIMP-2, in which the ten amino acid residue sequence of APP-derived MMP-2 selective inhibitory peptide (APP-IP) is added to the N terminus of TIMP-2. The APP-IP and TIMP-2 regions of the fusion protein are designed to interact with the active site and the hemopexin-like domain of MMP-2, respectively. The reactive site of the TIMP-2 region, which has broad specificity against MMPs, is blocked by the APP-IP adduct. The recombinant APP-IP-TIMP-2 showed strong inhibitory activity toward MMP-2 (K_i^app = 0.68 pm), whereas its inhibitory activity toward MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, or MT1-MMP was six orders of magnitude or more weaker (IC₅₀ > 1 μm). The fusion protein inhibited the activation of pro-MMP-2 in the concanavalin A-stimulated HT1080 cells, degradation of type IV collagen by the cells, and the migration of stimulated cells. Compared with the decapeptide APP-IP (t_½ = 30 min), APP-IP-TIMP-2 (t_½ ≫ 96 h) showed a much longer half-life in cultured tumor cells. Therefore, the fusion protein may be a useful tool to evaluate contributions of proteolytic activity of MMP-2 in various pathophysiological processes. It may also be developed as an effective anti-tumor drug with restricted side effects.     

Trp2313-His2315 of Factor VIII C2 Domain Is Involved in Membrane Binding: STRUCTURE OF A COMPLEX BETWEEN THE C2 DOMAIN AND AN INHIBITOR OF MEMBRANE BINDING*

Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 Å) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed β-strand of the C2 domain, Trp²³¹³-His²³¹⁵. This result indicates that the Trp²³¹³-His²³¹⁵ segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.     

The Crystal Structure of the Active Domain of Anopheles Anti-platelet Protein,a Powerful Anti-coagulant,in Complex with an Antibody

Blood clotting is a vitally important process that must be carefully regulated to prevent blood loss on one hand and thrombosis on the other. Severe injury and hemophilia may be treated with pro-coagulants, whereas risk of obstructive clotting or embolism may be reduced with anti-coagulants. Anti-coagulants are an extremely important class of drug, one of the most widely used types of medication, but there remains a pressing need for novel treatments, however, as present drugs such as warfarin have significant drawbacks. Nature provides a number of examples of anti-coagulant proteins produced by blood-sucking animals, which may provide templates for the development of new small molecules with similar physiological effects. We have, therefore, studied an Anopheles anti-platelet protein from a malaria vector mosquito and report its crystal structure in complex with an antibody. Overall the protein is extremely sensitive to proteolysis, but the crystal structure reveals a stable domain built from two helices and a turn, which corresponds to the functional region. The antibody raised against Anopheles anti-platelet protein prevents it from binding collagen. Our work, therefore, opens new avenues to the development of both novel small molecule anti-clotting agents and anti-malarials.