Molecular basis of the interaction between complement receptor type 2 (CR2/CD21) and Epstein-Barr virus glycoprotein gp350

The binding of the Epstein-Barr virus glycoprotein gp350 by complement receptor type 2 (CR2) is critical for viral attachment to B lymphocytes. We set out to test hypotheses regarding the molecular nature of this interaction by developing an enzyme-linked immunosorbent assay (ELISA) for the efficient analysis of the gp350-CR2 interaction by utilizing wild-type and mutant forms of recombinant gp350 and also of the CR2 N-terminal domains SCR1 and SCR2 (designated CR2 SCR1-2). To delineate the CR2-binding site on gp350, we generated 17 gp350 single-site substitutions targeting an area of gp350 that has been broadly implicated in the binding of both CR2 and the major inhibitory anti-gp350 monoclonal antibody (MAb) 72A1. These site-directed mutations identified a novel negatively charged CR2-binding surface described by residues Glu-21, Asp-22, Glu-155, Asp-208, Glu-210, and Asp-296. We also identified gp350 amino acid residues involved in non-charge-dependent interactions with CR2, including Tyr-151, Ile-160, and Trp-162. These data were supported by experiments in which phycoerythrin-conjugated wild-type and mutant forms of gp350 were incubated with CR2-expressing K562 cells and binding was assessed by flow cytometry. The ELISA was further utilized to identify several positively charged residues (Arg-13, Arg-28, Arg-36, Lys-41, Lys-57, Lys-67, Arg-83, and Arg-89) within SCR1-2 of CR2 that are involved in the binding interaction with gp350. These experiments allowed a comparison of those CR2 residues that are important for binding gp350 to those that define the epitope for an effective inhibitory anti-CR2 MAb, 171 (Asn-11, Arg-13, Ser-32, Thr-34, Arg-36, and Tyr-64). The mutagenesis data were used to calculate a model of the CR2-gp350 complex using the soft-docking program HADDOCK.     

Probing the molecular basis for potent and selective protein-tyrosine phosphatase 1B inhibition

Protein-tyrosine phosphatases (PTPs) are important for the control of proper cellular tyrosine phosphorylation. Despite the large number of PTPs encoded in the human genome and the emerging roles played by PTPs in human diseases, a detailed understanding of the role played by PTPs in normal physiology and in pathogenic conditions has been hampered by the absence of PTP-specific inhibitors. Such inhibitors could serve as useful tools for determining the physiological functions of PTPs and may constitute valuable therapeutics in the treatment of several human diseases. However, because of the highly conserved nature of the active site, it has been difficult to develop selective PTP inhibitors. By taking an approach to tether together two small ligands that can interact simultaneously with the active site and a unique proximal noncatalytic site, we have recently acquired Compound 2 (see Fig. 1), the most potent and selective PTP1B inhibitor identified to date, which exhibits several orders of magnitude selectivity in favor of PTP1B against a panel of PTPs. We describe an evaluation of the interaction between 2 and its analogs with PTP1B and its site-directed mutants selected based on hydrogen/deuterium exchange of PTP1B backbone amides in the presence and absence of 2. We have established the binding mode of Compound 2 and identified 12 PTP1B residues that are important for the potency and selectivity of Compound 2. Although many of the residues important for Compound 2 binding are not unique to PTP1B, the combinations of all contact residues differ between PTP isozymes, which suggest that the binding surface defined by these residues in individual PTPs determines inhibitor selectivity. Our results provide structural information toward understanding of the molecular basis for potent and selective PTP1B inhibition and further establish the feasibility of acquiring potent, yet highly selective, PTP inhibitory agents.     

Thermodynamic study of ligand binding to protein-tyrosine phosphatase 1B and its substrate-trapping mutants

The binding of several phosphonodifluoromethyl phenylalanine (F(2)Pmp)-containing peptides to protein-tyrosine phosphatase 1B (PTP1B) and its substrate-trapping mutants (C215S and D181A) has been studied using isothermal titration calorimetry. The binding of a high affinity ligand, Ac-Asp-Ala-Asp-Glu-F(2)Pmp-Leu-NH(2), to PTP1B (K(d) = 0.24 microm) is favored by both enthalpic and entropic contributions. Disruption of ionic interactions between the side chain of Arg-47 and the N-terminal acidic residues reduces the binding affinity primarily through the reduction of the TDeltaS term. The role of Arg-47 may be to maximize surface contact between PTP1B and the peptide, which contributes to high affinity binding. The active site Cys-215 --> Ser mutant PTP1B binds ligands with the same affinity as the wild-type enzyme. However, unlike wild-type PTP1B, peptide binding to C215S is predominantly driven by enthalpy change, which likely results from the elimination of the electrostatic repulsion between the thiolate anion and the phosphonate group. The increased enthalpic contribution is offset by reduction in the binding entropy, which may be the result of increased entropy of the unbound protein caused by this mutation. The general acid-deficient mutant D181A binds the peptide 5-fold tighter than the C215S mutant, consistent with the observation that the Asp to Ala mutant is a better "substrate-trapping" reagent than C215S. The increased binding affinity for D181A as compared with the wild-type PTP1B results primarily from an increase in the DeltaH of binding in the mutant, which may be related to decreased electrostatic repulsion between the phosphate moiety and PTP1B. These results have important implications for the design of high affinity PTP1B inhibitors.     

A bipartite mechanism for ERK2 recognition by its cognate regulators and substrates

Mitogen-activated protein (MAP) kinases control gene expression in response to extracellular stimuli and exhibit exquisite specificity for their cognate regulators and substrates. We performed a structure-based mutational analysis of ERK2 to identify surface areas that are important for recognition of its interacting proteins. We show that binding and activation of MKP3 by ERK2 involve two distinct protein-protein interaction sites in ERK2. Thus, the common docking (CD) site composed of Glu-79, Tyr-126, Arg-133, Asp-160, Tyr-314, Asp-316, and Asp-319 are important for high affinity MKP3 binding but not essential for ERK2-induced MKP3 activation. MKP3 activation requires residues Tyr-111, Thr-116, Leu-119, Lys-149, Arg-189, Trp-190, Glu-218, Arg-223, Lys-229, and His-230 in the ERK2 substrate-binding region, located distal to the common docking site. Interestingly, many of the residues important for MKP3 recognition are also used for Elk1 binding and phosphorylation. In addition to the shared residues, there are also residues that are unique to each target recognition. There is evidence indicating that the CD site and the substrate-binding region defined here are also utilized for MEK1 recognition, and indeed, we demonstrate that the binding of MKP3, Elk1, and MEK1 to ERK2 is mutually exclusive. Taken together, our data suggest that the efficiency and fidelity of ERK2 signaling is achieved by a bipartite recognition process. In this model, one part of the ERK2-binding proteins (e.g. the kinase interaction motif sequence) docks to the CD site located on the back side of the ERK2 catalytic pocket for high affinity association, whereas the interaction of the substrate-binding region with another structural element (e.g. the FXFP motif in MKP3 and Elk1) may not only stabilize binding but also provide contacts crucial for modulating the activity and/or specificity of ERK2 target molecules.     

Charged amino acids of the N-terminal domain are involved in coupling binding and gating in alpha7 nicotinic receptors

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of conformational changes resulting in channel opening. Previously, we have shown that the aspartate residue Asp-266 at the M2-M3 linker of the alpha7 nicotinic receptor is involved in connecting binding and gating. High resolution structural data suggest that this region could interact with the so-called loops 2 and 7 of the extracellular N-terminal region. In this case, certain charged amino acids present in these loops could integrate together with Asp-266 and other amino acids, a mechanism involved in channel activation. To test this hypothesis, all charged residues in these loops, Asp-42, Asp-44, Glu-45, Lys-46, Asp-128, Arg-130, and Asp-135, were substituted with other amino acids, and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Glu-45, Lys-46, and Asp-135 exhibited poor or null functional responses to different nicotinic agonists regardless of significant membrane expression, whereas D128A showed a gain of function effect. Because the double reverse charge mutant K46D/D266K did not restore receptor function, a gating mechanism controlled by the pairwise electrostatic interaction between these residues is not likely. Rather, a network of interactions formed by residues Lys-46, Asp-128, Asp-135, Asp-266, and possibly others appears to link agonist binding to channel gating.     

Structural basis of plasticity in protein tyrosine phosphatase 1B substrate recognition

Protein tyrosine phosphatase 1B (PTP1B) displays a preference for peptides containing acidic as well as aromatic/aliphatic residues immediately NH(2)-terminal to phosphotyrosine. The structure of PTP1B bound with DADEpYL-NH(2) (EGFR(988)(-)(993)) offers a structural explanation for PTP1B's preference for acidic residues [Jia, Z., Barford, D., Flint, A. J., and Tonks, N. K. (1995) Science 268, 1754-1758]. We report here the crystal structures of PTP1B in complex with Ac-ELEFpYMDYE-NH(2) (PTP1B.Con) and Ac-DAD(Bpa)pYLIPQQG (PTP1B.Bpa) determined to 1.8 and 1.9 A resolution, respectively. A structural analysis of PTP1B.Con and PTP1B.Bpa shows how aromatic/aliphatic residues at the -1 and -3 positions of peptide substrates are accommodated by PTP1B. A comparison of the structures of PTP1B.Con and PTP1B.Bpa with that of PTP1B.EGFR(988)(-)(993) reveals the structural basis for the plasticity of PTP1B substrate recognition. PTP1B is able to bind phosphopeptides by utilizing common interactions involving the aromatic ring and phosphate moiety of phosphotyrosine itself, two conserved hydrogen bonds between the Asp48 carboxylate side chain and the main chain nitrogens of the pTyr and residue 1, and a third between the main chain nitrogen of Arg47 and the main chain carbonyl of residue -2. The ability of PTP1B to accommodate both acidic and hydrophobic residues immediately NH(2)-terminal to pTyr appears to be conferred upon PTP1B by a single residue, Arg47. Depending on the nature of the NH(2)-terminal amino acids, the side chain of Arg47 can adopt one of two different conformations, generating two sets of distinct peptide binding surfaces. When an acidic residue is positioned at position -1, a preference for a second acidic residue is also observed at position -2. However, when a large hydrophobic group occupies position -1, Arg47 adopts a new conformation so that it can participate in hydrophobic interactions with both positions -1 and -3.     

Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA)

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.     

Molecular determinants of receptor binding and signaling by the CX3C chemokine fractalkine.

Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors.     

Functional mapping of charged residues of the 82-116 sequence in factor Xa: evidence that lysine 96 is a factor Va independent recognition site for prothrombin in the prothrombinase complex

It has been hypothesized that two antiparallel structures comprised of residues 82-91 and 102-116 in factor Xa (fXa) may harbor a factor Va- (fVa-) dependent prothrombin recognition site in the prothrombinase complex. There are 11 charged residues in the 82-116 loop of human fXa (Glu-84, Glu-86, Lys-90, Arg-93, Lys-96, Glu-97, Asp-100, Asp-102, Arg-107, Lys-109, and Arg-115). With the exception of Glu-84, which did not express, and Asp-102, which is a catalytic residue, we expressed the Ala substitution mutants of all other residues and evaluated their proteolytic and amidolytic activities in both the absence and presence of fVa. K96A and K109A activated prothrombin with 5-10-fold impaired catalytic efficiency in the absence of fVa. All mutants, however, exhibited normal activity toward the substrate in the presence of fVa. K109A also exhibited impaired amidolytic activity and affinity for Na(+); however, both fVa and higher Na(+) restored the catalytic defect caused by the mutation. Analysis of the X-ray crystal structure of fXa indicated that Glu-84 may interact by a salt bridge with Lys-109, explaining the lack of expression of E84A and the lower activity of K109A in the absence of fVa. These results suggest that none of the residues under study is a fVa-dependent recognition site for prothrombin in the prothrombinase complex; however, Lys-96 is a recognition site for the substrate independent of the cofactor. Moreover, the 82-116 loop is energetically linked to fVa and Na(+) binding sites of the protease.     

Characterization of the stromal cell-derived factor-1alpha-heparin complex

The binding of chemokines to glycosaminoglycans is thought to play a crucial role in chemokine functions. It has recently been shown that stromal cell-derived factor-1alpha (SDF-1alpha), a CXC chemokine with potent anti-human immunodeficiency virus activity, binds to heparan sulfate through a typical consensus sequence for heparin recognition (BBXB, where B is a basic residue KHLK, amino acids 24-27). Calculation of the accessible surface, together with the electrostatic potential of the SDF-1alpha dimer, revealed that other amino acids (Arg-41 and Lys-43) are found in the same surface area and contribute to the creation of a positively charged crevice, located at the dimer interface. GRID calculations confirmed that this binding site will be the most energetically favored area for the interaction with sulfate groups. Site-directed mutagenesis and surface plasmon resonance-based binding assays were used to investigate the structural basis for SDF-1alpha binding to heparin. Among the residues clustered in this basic surface area, Lys-24 and Lys-27 have dominant roles and are essential for interaction with heparin. Amino acids Arg-41 and Lys-43 participate in the binding but are not strictly required for the interaction to take place. Direct binding assays and competition analysis with monoclonal antibodies also permitted us to show that the N-terminal residue (Lys-1), an amino acid critical for receptor activation, is involved in complex formation. Binding studies with selectively desulfated heparin, heparin oligosaccharides, and heparitinase-resistant heparan sulfate fragments showed that a minimum size of 12-14 monosaccharide units is required for efficient binding and that 2-O- and N-sulfate groups have a dominant role in the interaction. Finally, the heparin-binding site was identified on the crystal structure of SDF-1alpha, and a docking study was undertaken. During the energy minimization process, heparin lost its perfect ribbon shape and fitted the protein surface perfectly. In the model, Lys-1, Lys-24, Lys-27, and Arg-41 were found to have the major role in binding a polysaccharide fragment consisting of 13 monosaccharide units.     

Crystal structure and mutational analysis of heparan sulfate 3-O-sulfotransferase isoform 1

Heparan sulfate interacts with antithrombin, a protease inhibitor, to regulate blood coagulation. Heparan sulfate 3-O-sulfotransferase isoform 1 performs the crucial last step modification in the biosynthesis of anticoagulant heparan sulfate. This enzyme transfers the sulfuryl group (SO(3)) from 3'-phosphoadenosine 5'-phosphosulfate to the 3-OH position of a glucosamine residue to form the 3-O-sulfo glucosamine, a structural motif critical for binding of heparan sulfate to antithrombin. In this study, we report the crystal structure of 3-O-sulfotransferase isoform 1 at 2.5-A resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate. This structure reveals residues critical for 3'-phosphoadenosine 5'-phosphosulfate binding and suggests residues required for the binding of heparan sulfate. In addition, site-directed mutagenesis analyses suggest that residues Arg-67, Lys-68, Arg-72, Glu-90, His-92, Asp-95, Lys-123, and Arg-276 are essential for enzymatic activity. Among these essential amino acid residues, we find that residues Arg-67, Arg-72, His-92, and Asp-95 are conserved in heparan sulfate 3-O-sulfotransferases but not in heparan N-deacetylase/N-sulfotransferase, suggesting a role for these residues in conferring substrate specificity. Results from this study provide information essential for understanding the biosynthesis of anticoagulant heparan sulfate and the general mechanism of action of heparan sulfate sulfotransferases.     

The active site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis

Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K(m) and V(max)) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta- and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.     

Crystal structure of the Yersinia protein-tyrosine phosphatase YopH complexed with a specific small molecule inhibitor

The pathogenic bacteria Yersinia are causative agents in human diseases ranging from gastrointestinal syndromes to bubonic plague. There is increasing risk of misuse of infectious agents, such as Yersinia pestis, as weapons of terror as well as instruments of warfare for mass destruction. Because the phosphatase activity of the Yersinia protein tyrosine phosphatase, YopH, is essential for virulence in the Yersinia pathogen, potent and selective YopH inhibitors are expected to serve as novel anti-plague agents. We have identified a specific YopH small molecule inhibitor, p-nitrocatechol sulfate (pNCS), which exhibits a Ki value of 25 microM for YopH and displays a 13-60-fold selectivity in favor of YopH against a panel of mammalian PTPs. To facilitate the understanding of the underlying molecular basis for tight binding and specificity, we have determined the crystal structure of YopH in complex with pNCS at a 2.0-A resolution. The structural data are corroborated by results from kinetic analyses of the interactions of YopH and its site-directed mutants with pNCS. The results show that while the interactions of the sulfuryl moiety and the phenyl ring with the YopH active site contribute to pNCS binding affinity, additional interactions of the hydroxyl and nitro groups in pNCS with Asp-356, Gln-357, Arg-404, and Gln-446 are responsible for the increased potency and selectivity. In particular, we note that residues Arg-404, Glu-290, Asp-356, and a bound water (WAT185) participate in a unique H-bonding network with the hydroxyl group ortho to the sulfuryl moiety, which may be exploited to design more potent and specific YopH inhibitors.     

Identification of amino acid residues essential for the catalytic reaction of Bacillus kaustophilus leucine aminopeptidase

The functional significance of amino acid residues Lys-265, Asp-270, Lys-277, Asp-288, Asp-347, Glu-349, and Arg-351 of Bacillus kaustophilus leucine aminopeptidase was explored by site-directed mutagenesis. Variants with an apparent molecular mass of approximately 54 kDa were overexpressed in Escherichia coli and purified to homogeneity by nickel-chelate chromatography. The purified mutant enzymes had no LAP activity, implying that these residues are important for the catalytic reaction of the enzyme.     

The structure of PTP-1B in complex with a peptide inhibitor reveals an alternative binding mode for bisphosphonates

Inhibitors of PTP-1B could be therapeutically beneficial in the treatment of type 2 diabetes. Owing to the large number of phosphatases in the cell, inhibitors against PTP-1B must not only be potent but selective as well. N-Benzoyl-L-glutamyl-[4-phosphono(difluoromethyl)]-L-phenylalanine-[4-phosphono(difluoro-methyl)]-L-phenylalanineamide (BzN-EJJ-amide) is a low nanomolar inhibitor of PTP-1B that shows selectivity over several protein tyrosine phosphatases. To gain an insight into the basis of its potency and selectivity, we evaluated several analogues of the inhibitor and introduced amino acid substitutions into PTP-1B by site-directed mutagenesis. We also determined the crystal structure of PTP-1B in complex with BzN-EJJ-amide at 2.5 A resolution. Our results indicate that the high inhibitory potency is due to interactions of several of its chemical groups with specific protein residues. An interaction between BzN-EJJ-amide and Asp48 is of particular significance, as substitution of Asp48 to alanine resulted in a 100-fold loss in potency. The crystal structure also revealed an unexpected binding orientation for a bisphosphonate inhibitor on PTP-1B, where the second difluorophosphonomethyl phenylalanine (F(2)PMP) moiety is bound close to Arg47 rather than in the previously identified second aryl phosphate site demarked by Arg24 and Arg254. Our results suggest that potent and selective PTP-1B inhibitors may be designed by targeting the region containing Arg47 and Asp48.     

Molecular modeling of protein tyrosine phosphatase 1B (PTP 1B) inhibitors

Binding modes of a series of aryloxymethylphosphonates and monoanionic biosteres of phosphate group from a series of benzylic alpha,alpha-diflluoro phosphate and its biosteres as protein tyrosine phosphatase 1B (PTP 1B) inhibitors have been identified by molecular modeling techniques. We have performed docking and molecular dynamics simulations of these inhibitors with PTP 1B enzyme. The initial conformation of the inhibitors for docking was obtained from simulated annealing technique. Solvent accessible surface area calculations suggested that active site of PTP 1B is highly hydrophobic. The results indicate that for aryloxymethylphosphonates, in addition to hydrogen bonding interactions, Tyr46, Arg47, Asp48, Val49, Glu115, Lys116, Lys120 amino acid residues of PTP 1B are responsible for governing inhibitor potency of the compounds. The sulfonate and tetrazole functional groups have been identified as effective monoanionic biosteres of phosphate group and biphenyl ring system due to its favorable interactions with Glu115, Lys116, Lys120 residues of PTP 1B found to be more suitable aromatic functionality than naphthalene ring system for benzylic alpha,alpha-diflluoro phosphate and its biosteres. The information generated from the present study should be useful in the design of more potent PTP 1B inhibitors as anti diabetic agents.     

Crystal structure of Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in complex with trypsin. Implications for KD1 specificity of inhibition

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 inhibits trypsin, plasmin, and factor VIIa (FVIIa)/tissue factor with Ki values of 13, 3, and 1640 nM, respectively. To investigate the molecular specificity of KD1, crystals of the complex of KD1 with bovine beta-trypsin were obtained that diffracted to 1.8 A. The P1 residue Arg-15 (bovine pancreatic trypsin inhibitor numbering) in KD1 interacts with Asp-189 (chymotrypsin numbering) and with the carbonyl oxygens of Gly-219 and Ogamma of Ser-190. Leu-17, Leu-18, Leu-19, and Leu-34 in KD1 make van der Waals contacts with Tyr-39, Phe-41, and Tyr-151 in trypsin, forming a hydrophobic interface. Molecular modeling indicates that this complementary hydrophobic patch is composed of Phe-37, Met-39, and Phe-41 in plasmin, whereas in FVIIa/tissue factor, it is essentially absent. Arg-20, Tyr-46, and Glu-39 in KD1 interact with trypsin through ordered water molecules. In contrast, insertions in the 60-loop in plasmin and FVIIa allow Arg-20 of KD1 to directly interact with Glu-60 in plasmin and Asp-60 in FVIIa. Moreover, Tyr-46 in KD1 electrostatically interacts with Lys-60A and Arg-60D in plasmin and Lys-60A in FVIIa. Glu-39 in KD1 interacts directly with Arg-175 of the basic patch in plasmin, whereas in FVIIa, such interactions are not possible. Thus, the specificity of KD1 for plasmin is attributable to hydrophobic and direct electrostatic interactions. For trypsin, hydrophobic interactions are intact, and electrostatic interactions are weak, whereas for FVIIa, hydrophobic interactions are missing, and electrostatic interactions are partially intact. These findings provide insight into the protease selectivity of KD1.     

Biochemical characterization of the chondroitinase B active site

Chondroitinase B from Flavobacterium heparinum is the only known lyase that cleaves the glycosaminoglycan, dermatan sulfate (DS), as its sole substrate. A recent co-crystal structure of chondroitinase B with a disaccharide product of DS depolymerization has provided some insight into the location of the active site and suggested potential roles of some active site residues in substrate binding and catalysis. However, this co-crystal structure was not representative of the actual enzyme-substrate complex, because the disaccharide product did not have the right length or the chemical structure of the minimal substrate (tetrasaccharide) involved in catalysis. Therefore, only a limited picture of the functional role of active site residues in DS depolymerization was presented in previous structural studies. In this study, by docking a DS tetrasaccharide into the proposed active site of the enzyme, we have identified novel roles of specific active site amino acids in the catalytic function of chondroitinase B. Our conformational analysis also revealed a unique, symmetrical arrangement of active site amino acids that may impinge on the catalytic mechanism of action of chondroitinase B. The catalytic residues Lys-250, Arg-271, His-272, and Glu-333 along with the substrate binding residues Arg-363 and Arg-364 were mutated using site-directed mutagenesis, and the kinetics and product profile of each mutant were compared with recombinant chondroitinase B. Mutating Lys-250 to alanine resulted in inactivation of the enzyme, potentially attributable to the role of the residue in stabilizing the carbanion intermediate formed during enzymatic catalysis. The His-272 and Glu-333 mutants showed diminished enzymatic activity that could be indicative of a possible role for one or both residues in the abstraction of the C-5 proton from the galactosamine. In addition, the Arg-364 mutant had an altered product profile after exhaustive digestion of DS, suggesting a role for this residue in defining the substrate specificity of chondroitinase B.     

New binding site on common molecular scaffold provides HERG channel specificity of scorpion toxin BeKm-1

The scorpion toxin BeKm-1 is unique among a variety of known short scorpion toxins affecting potassium channels in its selective action on ether-a-go-go-related gene (ERG)-type channels. BeKm-1 shares the common molecular scaffold with other short scorpion toxins. The toxin spatial structure resolved by NMR consists of a short alpha-helix and a triple-stranded antiparallel beta-sheet. By toxin mutagenesis study we identified the residues that are important for the binding of BeKm-1 to the human ERG K+ (HERG) channel. The most critical residues (Tyr-11, Lys-18, Arg-20, Lys-23) are located in the alpha-helix and following loop whereas the "traditional" functional site of other short scorpion toxins is formed by residues from the beta-sheet. Thus the unique location of the binding site of BeKm-1 provides its specificity toward the HERG channel.