Engineering kunitz domain 1 (KD1) of human tissue factor pathway inhibitor-2 to selectively inhibit fibrinolysis: properties of KD1-L17R variant

Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2' position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2' residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with ～6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was ～8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R.     

Alboserpin, a factor Xa inhibitor from the mosquito vector of yellow fever, binds heparin and membrane phospholipids and exhibits antithrombotic activity

The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) ~ 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.     

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.     

Structural basis for selective small molecule kinase inhibition of activated c-Met

The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix αC and the G loop to generate a viable active site. Helix αC adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.     

Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

The activation of antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reactions by 300- to 500-fold. Relative to FXa, AT inhibits FIXa with ∼40-fold slower rate constant. Structural data suggest that differences in the residues of the 39-loop (residues 31–41) may partly be responsible for the differential reactivity of the two proteases with AT. This loop is highly acidic in FXa, containing three Glu residues at positions 36, 37, and 39. By contrast, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. To determine whether differences in the residues of this loop contribute to the slower reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which the 39-loop of the protease was replaced with the corresponding loop of FXa. The chimeric mutant cleaved a FIXa-specific chromogenic substrate with normal catalytic efficiency, however, the mutant exhibited ∼5-fold enhanced reactivity with AT specifically in the absence of the cofactor, heparin. Further studies revealed that the FIXa mutant activates factor X with ∼4-fold decreased k_cat and ∼2-fold decreased K_m, although the mutant interacted normally with factor VIIIa. Based on these results we conclude that residues of the 39-loop regulate the cofactor-independent interaction of FIXa with its physiological inhibitor AT and substrate factor X.     

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.     

Structural study of the complex stereoselectivity of human butyrylcholinesterase for the neurotoxic V-agents

Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous time-dependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs. The stereoselectivity of BChE is an important parameter for its efficiency at scavenging the most toxic OPs enantiomer for AChE. Crystals of BChE inhibited in solution or in cristallo with racemic V-agents (VX, Russian VX, and Chinese VX) systematically show the formation of the P(S) adduct. In this configuration, no catalysis of aging seems possible as confirmed by the three-dimensional structures of the three conjugates incubated over a period exceeding a week. Crystals of BChE soaked in optically pure VX(R)-(+) and VX(S)-(-) solutions lead to the formation of the P(S) and P(R) adduct, respectively. These structural data support an in-line phosphonylation mechanism. Additionally, they show that BChE reacts with VX(R)-(+) in the presence of racemic mixture of V-agents, at odds with earlier kinetic results showing a moderate higher inhibition rate for VX(S)-(-). These combined results suggest that the simultaneous presence of both enantiomers alters the enzyme stereoselectivity. In summary, the three-dimensional data show that BChE reacts preferentially with P(R) enantiomer of V-agents and does not age, in complete contrast to AChE, which is selectively inhibited by the P(S) enantiomer and ages.     

Basis for the Specificity and Activation of the Serpin Protein Z-dependent Proteinase Inhibitor (ZPI) as an Inhibitor of Membrane-associated Factor Xa

The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction ∼2000-fold in the presence of phospholipid and Ca²⁺. To elucidate the role of PZ, we determined the x-ray structure of Gla-domainless PZ (PZ_ΔGD) complexed with protein Z-dependent proteinase inhibitor (ZPI). The PZ pseudocatalytic domain bound ZPI at a novel site through ionic and polar interactions. Mutation of four ZPI contact residues eliminated PZ binding and membrane-dependent PZ acceleration of fXa inhibition. Modeling of the ternary Michaelis complex implicated ZPI residues Glu-313 and Glu-383 in fXa binding. Mutagenesis established that only Glu-313 is important, contributing ∼5–10-fold to rate acceleration of fXa and fXIa inhibition. Limited conformational change in ZPI resulted from PZ binding, which contributed only ∼2-fold to rate enhancement. Instead, template bridging from membrane association, together with previously demonstrated interaction of the fXa and ZPI Gla domains, resulted in an additional ∼1000-fold rate enhancement. To understand why ZPI has P1 tyrosine, we examined a P1 Arg variant. This reacted at a diffusion-limited rate with fXa, even without PZ, and predominantly as substrate, reflecting both rapid acylation and deacylation. P1 tyrosine thus ensures that reaction with fXa or most other arginine-specific proteinases is insignificant unless PZ binds and localizes ZPI and fXa on the membrane, where the combined effects of Gla-Gla interaction, template bridging, and interaction of fXa with Glu-313 overcome the unfavorability of P1 Tyr and ensure a high rate of reaction as an inhibitor.     

The replication focus targeting sequence (RFTS) domain is a DNA-competitive inhibitor of Dnmt1

Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for maintenance of cytosine methylation at CpG dinucleotides in the mammalian genome. The N-terminal replication focus targeting sequence (RFTS) domain of Dnmt1 has been implicated in subcellular localization, protein association, and catalytic function. However, progress in understanding its function has been limited by the lack of assays for and a structure of this domain. Here, we show that the naked DNA- and polynucleosome-binding activities of Dnmt1 are inhibited by the RFTS domain, which functions by virtue of binding the catalytic domain to the exclusion of DNA. Kinetic analysis with a fluorogenic DNA substrate established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity. The crystal structure of the RFTS domain reveals a novel fold and supports a mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may activate Dnmt1 for DNA binding.     

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.     

Structures of human Golgi-resident glutaminyl cyclase and its complexes with inhibitors reveal a large loop movement upon inhibitor binding

Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05–1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC.     

Crystal Structure of the Protealysin Precursor: INSIGHTS INTO PROPEPTIDE FUNCTION*

Protealysin (PLN) belongs to the M4 family of peptidases that are commonly known as thermolysin-like proteases (TLPs). All TLPs are synthesized as precursors containing N-terminal propeptides. According to the primary structure of the N-terminal propeptides, the family is divided into two distinct groups. Representatives of the first group including thermolysin and all TLPs with known three-dimensional structures have long prosequences (∼200 amino acids). Enzymes of the second group, whose prototype is protealysin, have short (∼50 amino acids) propeptides. Here, we present the 1.8 Å crystal structure of PLN precursor (proPLN), which is the first three-dimensional structure of a TLP precursor. Whereas the structure of the catalytic domain of proPLN is similar overall to previously reported structures of mature TLPs, it has specific features, including the absence of calcium-binding sites, and different structures of the N-terminal region and substrate-binding site. PLN propeptide forms a separate domain in the precursor and likely acts as an inhibitor that blocks the substrate-binding site and fixes the “open” conformation of the active site, which is unfavorable for catalysis. Furthermore the conserved PPL motif identified in our previous studies directly interacts with the S′ subsites of the active center being a critical element of the propeptide-catalytic domain interface. Comparison of the primary structures of TLPs with short propeptides suggests that the specific features revealed in the proPLN crystal structure are typical for all protealysin-like enzymes. Thus, such proteins can be considered as a separate subfamily of TLPs.     

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.     

Molecular recognition of chymotrypsin by the serine protease inhibitor ecotin from Yersinia pestis

Resistance to antibiotics is a problem not only in terms of healthcare but also biodefense. Engineering of resistance into a human pathogen could create an untreatable biothreat pathogen. One such pathogen is Yersinia pestis, the causative agent of plague. Previously, we have used a bioinformatic approach to identify proteins that may be suitable targets for antimicrobial therapy and in particular for the treatment of plague. The serine protease inhibitor ecotin was identified as one such target. We have carried out mutational analyses in the closely related Yersinia pseudotuberculosis, validating that the ecotin gene is a virulence-associated gene in this bacterium. Y. pestis ecotin inhibits chymotrypsin. Here, we present the structure of ecotin in complex with chymotrypsin to 2.74 Å resolution. The structure features a biologically relevant tetramer whereby an ecotin dimer binds to two chymotrypsin molecules, similar to what was observed in related serine protease inhibitor structures. However, the vast majority of the interactions in the present structure are distinctive, indicating that the broad specificity of the inhibitor for these proteases is based largely on its capacity to recognize features unique to each of them. These findings will have implications for the development of small ecotin inhibitors for therapeutic use.     

Structural and enzymatic insights into caspase-2 protein substrate recognition and catalysis

Caspase-2, the most evolutionarily conserved member in the human caspase family, may play important roles in stress-induced apoptosis, cell cycle regulation, and tumor suppression. In biochemical assays, caspase-2 uniquely prefers a pentapeptide (such as VDVAD) rather than a tetrapeptide, as required for efficient cleavage by other caspases. We investigated the molecular basis for pentapeptide specificity using peptide analog inhibitors and substrates that vary at the P5 position. We determined the crystal structures of apo caspase-2, caspase-2 in complex with peptide inhibitors VDVAD-CHO, ADVAD-CHO, and DVAD-CHO, and a T380A mutant of caspase-2 in complex with VDVAD-CHO. Two residues, Thr-380 and Tyr-420, are identified to be critical for the P5 residue recognition; mutation of the two residues reduces the catalytic efficiency by about 4- and 40-fold, respectively. The structures also provide a series of snapshots of caspase-2 in different catalytic states, shedding light on the mechanism of capase-2 activation, substrate binding, and catalysis. By comparing the apo and inhibited caspase-2 structures, we propose that the disruption of a non-conserved salt bridge between Glu-217 and the invariant Arg-378 is important for the activation of caspase-2. These findings broaden our understanding of caspase-2 substrate specificity and catalysis.     

Selective inhibition of biotin protein ligase from Staphylococcus aureus

There is a well documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy to combat resistance is to discover new chemical classes immune to current resistance mechanisms that inhibit essential metabolic enzymes. Many of the obvious drug targets that have no homologous isozyme in the human host have now been investigated. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme over the human homolog. We have demonstrated that the essential enzyme biotin protein ligase (BPL) from the clinically important pathogen Staphylococcus aureus could be selectively inhibited. Linking biotin to adenosine via a 1,2,3 triazole yielded the first BPL inhibitor selective for S. aureus BPL over the human equivalent. The synthesis of new biotin 1,2,3-triazole analogues using click chemistry yielded our most potent structure (K(i) 90 nM) with a >1100-fold selectivity for the S. aureus BPL over the human homologue. X-ray crystallography confirmed the mechanism of inhibitor binding. Importantly, the inhibitor showed cytotoxicity against S. aureus but not cultured mammalian cells. The biotin 1,2,3-triazole provides a novel pharmacophore for future medicinal chemistry programs to develop this new antibiotic class.     

Structure and Function of Human Xylulokinase,an Enzyme with Important Roles in Carbohydrate Metabolism

d-Xylulokinase (XK; EC 2.7.1.17) catalyzes the ATP-dependent phosphorylation of d-xylulose (Xu) to produce xylulose 5-phosphate (Xu5P). In mammals, XK is the last enzyme in the glucuronate-xylulose pathway, active in the liver and kidneys, and is linked through its product Xu5P to the pentose-phosphate pathway. XK may play an important role in metabolic disease, given that Xu5P is a key regulator of glucose metabolism and lipogenesis. We have expressed the product of a putative human XK gene and identified it as the authentic human d-xylulokinase (hXK). NMR studies with a variety of sugars showed that hXK acts only on d-xylulose, and a coupled photometric assay established its key kinetic parameters as K_m(Xu) = 24 ± 3 μm and k_cat = 35 ± 5 s⁻¹. Crystal structures were determined for hXK, on its own and in complexes with Xu, ADP, and a fluorinated inhibitor. These reveal that hXK has a two-domain fold characteristic of the sugar kinase/hsp70/actin superfamily, with glycerol kinase as its closest relative. Xu binds to domain-I and ADP to domain-II, but in this open form of hXK they are 10 Å apart, implying that a large scale conformational change is required for catalysis. Xu binds in its linear keto-form, sandwiched between a Trp side chain and polar side chains that provide exquisite hydrogen bonding recognition. The hXK structure provides a basis for the design of specific inhibitors with which to probe its roles in sugar metabolism and metabolic disease.     

A novel heparin-dependent inhibitor of activated protein C that potentiates consumptive coagulopathy in Russell's viper envenomation

The activation of coagulation factors V and X by Russell's viper venom (RVV) has been implicated in the development of consumptive coagulopathies in severely envenomed patients. However, factor Va is prone to inactivation by activated protein C (APC), an important serine protease that negatively regulates blood coagulation. It is therefore hypothesized that APC may be down-regulated by some of the venom components. In this study, we managed to isolate a potent Kunitz-type APC inhibitor, named DrKIn-I. Using chromogenic substrate, DrKIn-I dose-dependently inhibited the activity of APC. Heparin potentiated the inhibition and reduced the IC(50) of DrKIn-I by 25-fold. DrKIn-I, together with heparin, also protected factor Va from APC-mediated inactivation. Using surface plasmon resonance, DrKIn-I exhibited fast binding kinetics with APC (association rate constant = 1.7 × 10(7) M(-1) s(-1)). Direct binding assays and kinetic studies revealed that this inhibition (K(i) = 53 pM) is due to the tight binding interactions of DrKIn-I with both heparin and APC. DrKIn-I also effectively reversed the anticoagulant activity of APC and completely restored the thrombin generation in APC-containing plasma. Furthermore, although the injection of either DrKIn-I or RVV-X (the venom factor X-activator) into ICR mice did not significantly deplete the plasma fibrinogen concentration, co-administration of DrKIn-I with RVV-X resulted in complete fibrinogen consumption and the deposition of fibrin thrombi in the glomerular capillaries. Our results provide new insights into the pathogenesis of RVV-induced coagulopathies and indicate that DrKIn-I is a novel APC inhibitor that is associated with potentially fatal thrombotic complications in Russell's viper envenomation.     

Structure and function of an arabinoxylan-specific xylanase

The enzymatic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The enzymes that catalyze this process include xylanases that degrade xylan, a β-1,4-xylose polymer that is decorated with various sugars. Although xylanases efficiently hydrolyze unsubstituted xylans, these enzymes are unable to access highly decorated forms of the polysaccharide, such as arabinoxylans that contain arabinofuranose decorations. Here, we show that a Clostridium thermocellum enzyme, designated CtXyl5A, hydrolyzes arabinoxylans but does not attack unsubstituted xylans. Analysis of the reaction products generated by CtXyl5A showed that all the oligosaccharides contain an O3 arabinose linked to the reducing end xylose. The crystal structure of the catalytic module (CtGH5) of CtXyl5A, appended to a family 6 noncatalytic carbohydrate-binding module (CtCBM6), showed that CtGH5 displays a canonical (α/β)(8)-barrel fold with the substrate binding cleft running along the surface of the protein. The catalytic apparatus is housed in the center of the cleft. Adjacent to the -1 subsite is a pocket that could accommodate an l-arabinofuranose-linked α-1,3 to the active site xylose, which is likely to function as a key specificity determinant. CtCBM6, which adopts a β-sandwich fold, recognizes the termini of xylo- and gluco-configured oligosaccharides, consistent with the pocket topology displayed by the ligand-binding site. In contrast to typical modular glycoside hydrolases, there is an extensive hydrophobic interface between CtGH5 and CtCBM6, and thus the two modules cannot function as independent entities.     

Crystal structure of novel metallocarboxypeptidase inhibitor from marine mollusk Nerita versicolor in complex with human carboxypeptidase A4

NvCI is a novel exogenous proteinaceous inhibitor of metallocarboxypeptidases from the marine snail Nerita versicolor. The complex between human carboxypeptidase A4 and NvCI has been crystallized and determined at 1.7 Å resolution. The NvCI structure defines a distinctive protein fold basically composed of a two-stranded antiparallel β-sheet connected by three loops and the inhibitory C-terminal tail and stabilized by three disulfide bridges. NvCI is a tight-binding inhibitor that interacts with the active site of the enzyme in a substrate-like manner. NvCI displays an extended and novel interface with human carboxypeptidase A4, responsible for inhibitory constants in the picomolar range for some members of the M14A subfamily of carboxypeptidases. This makes NvCI the strongest inhibitor reported so far for this family. The structural homology displayed by the C-terminal tails of different carboxypeptidase inhibitors represents a relevant example of convergent evolution.