Biomedical subjects

W Bode

Publications and source records attributed to W Bode.

At least 37 records · Page 2Linked to original sources

PubMed2001

Fine mapping of inhibitory anti-factor V antibodies using factor V C2 domain mutants. Identification of two antigenic epitopes involved in phospholipid binding.

Hemorrhagic factor V inhibitors frequently bind to the second C-type (C2) domain of factor V and interfere with phospholipid binding. To define specific residues recognized by inhibitors from four patients (one bovine thrombin-induced and three spontaneous antibodies), epitope mapping was performed using recombinant human factor V lacking most of the B-type domain (FV des B) and alanine-substituted mutants within the C2 domain (FV des B C2 mutants). FV des B C2 mutants located in the region between Lys2060 and Glu2069 were resistant to inhibition by three IgG preparations including the bovine thrombin-induced antibody in both prothrombinase and phospholipid-binding assays. In contrast, mutations at Lys2087 and Lys2092/Glu2096 were significantly resistant to inhibition by the fourth IgG preparation in both prothrombinase and phospholipid-binding assays. These results confirm interference of phospholipid binding by hemorrhagic factor V inhibitors and support the role(s) of these residues in phospholipid binding.

Aged↗

PubMed2000

Crystal structure of the human alpha-thrombin-haemadin complex: an exosite II-binding inhibitor.

The serine proteinase alpha-thrombin plays a pivotal role in the regulation of blood fluidity, and therefore constitutes a primary target in the treatment of various haemostatic disorders. Haemadin is a slow tight- binding thrombin inhibitor from the land-living leech Haemadipsa sylvestris. Here we present the 3.1 A crystal structure of the human alpha-thrombin- haemadin complex. The N-terminal segment of haemadin binds to the active site of thrombin, forming a parallel beta-strand with residues Ser214-Gly216 of the proteinase. This mode of binding is similar to that observed in another leech-derived inhibitor, hirudin. In contrast to hirudin, however, the markedly acidic C-terminal peptide of haemadin does not bind the fibrinogen-recognition exosite, but interacts with the heparin-binding exosite of thrombin. Thus, haemadin binds to thrombin according to a novel mechanism, despite an overall structural similarity with hirudin. Haemadin inhibits both free and thrombomodulin-bound alpha-thrombin, but not intermediate activation forms such as meizothrombin. This specific anticoagulant ability of haemadin makes it an ideal candidate for an antithrombotic agent, as well as a starting point for the design of novel antithrombotics.

Amino Acid Sequence↗

PubMed2000

(4-aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase.

Increased expression of the serine protease urokinase-type plasminogen activator (uPA) in tumor tissues is highly correlated with tumor cell migration, invasion, proliferation, progression, and metastasis. Thus inhibition of uPA activity represents a promising target for antimetastatic therapy. So far, only the x-ray crystal structure of uPA inactivated by H-Glu-Gly-Arg-chloromethylketone has been reported, thus limited data are available for a rational structure-based design of uPA inhibitors. Taking into account the trypsin-like arginine specificity of uPA, (4-aminomethyl)phenylguanidine was selected as a potential P1 residue and iterative derivatization of its amino group with various hydrophobic residues, and structure-activity relationship-based optimization of the spacer in terms of hydrogen bond acceptor/donor properties led to N-(1-adamantyl)-N'-(4-guanidinobenzyl)urea as a highly selective nonpeptidic uPA inhibitor. The x-ray crystal structure of the uPA B-chain complexed with this inhibitor revealed a surprising binding mode consisting of the expected insertion of the phenylguanidine moiety into the S1 pocket, but with the adamantyl residue protruding toward the hydrophobic S1' enzyme subsite, thus exposing the ureido group to hydrogen-bonding interactions. Although in this enzyme-bound state the inhibitor is crossing the active site, interactions with the catalytic residues Ser-195 and His-57 are not observed, but their side chains are spatially displaced for steric reasons. Compared with other trypsin-like serine proteases, the S2 and S3/S4 pockets of uPA are reduced in size because of the 99-insertion loop. Therefore, the peculiar binding mode of the new type of uPA inhibitors offers the possibility of exploiting optimized interactions at the S1'/S2' subsites to further enhance selectivity and potency. Because crystals of the uPA/benzamidine complex allow inhibitor exchange by soaking procedures, the structure-based design of new generations of uPA inhibitors can rely on the assistance of x-ray analysis.

Amino Acid Sequence↗

PubMed2000

Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex.

The serine proteinase alpha-thrombin causes blood clotting through proteolytic cleavage of fibrinogen and protease-activated receptors and amplifies its own generation by activating the essential clotting factors V and VIII. Thrombomodulin, a transmembrane thrombin receptor with six contiguous epidermal growth factor-like domains (TME1-6), profoundly alters the substrate specificity of thrombin from pro- to anticoagulant by activating protein C. Activated protein C then deactivates the coagulation cascade by degrading activated factors V and VIII. The thrombin-thrombomodulin complex inhibits fibrinolysis by activating the procarboxypeptidase thrombin-activatable fibrinolysis inhibitor. Here we present the 2.3 A crystal structure of human alpha-thrombin bound to the smallest thrombomodulin fragment required for full protein-C co-factor activity, TME456. The Y-shaped thrombomodulin fragment binds to thrombin's anion-binding exosite-I, preventing binding of procoagulant substrates. Thrombomodulin binding does not seem to induce marked allosteric structural rearrangements at the thrombin active site. Rather, docking of a protein C model to thrombin-TME456 indicates that TME45 may bind substrates in such a manner that their zymogen-activation cleavage sites are presented optimally to the unaltered thrombin active site.

Amino Acid Sequence↗

PubMed2000

The human mast cell tryptase tetramer: a fascinating riddle solved by structure.

Tryptases, the predominant proteins of human mast cells, have been implicated as pathogenetic mediators of allergic and inflammatory conditions, most notably asthma. Until recently, the fascinating properties that distinguish tryptases among the serine proteinases, particularly their activity as a heparin-stabilized tetramer, resistance to most proteinaceous inhibitors, and preference for peptidergic over macromolecular substrates presented a riddle. This review solves this riddle with the help of the crystal structure of the human beta(2)-tryptase tetramer, but also indicates controversies between the unique quaternary architecture and some experimental data.

Amino Acid Sequence↗

PubMed2000

Structural basis of the endoproteinase-protein inhibitor interaction.

Proteolytic enzymes are potentially hazardous to their protein environment, so that their activity must be carefully controlled. Living organisms use protein inhibitors as a major tool to regulate the proteolytic activity of proteinases. Most of the inhibitors for which 3D structures are available are directed towards serine proteinases, interacting with the active sites in a 'canonical' i.e. substrate-like manner via an exposed reactive site loop of conserved conformation. More recently, some non-canonically binding serine proteinase inhibitors directed against coagulation factors, in particular thrombin, a few cysteine proteinase inhibitors inhibitory towards papain-like proteinases, and three zinc endopeptidase inhibitors directed against metzincins and thermolysin have been characterised in the free and complexed state, displaying novel mechanisms of inhibition with their target proteinases. These different interaction modes are presented and briefly discussed with respect to the different strategies applied by nature.

Animals↗

PubMed2000

Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis.

We have previously determined that the C2-domain of human factor V (residues 2037-2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. Naturally occurring factor V inhibitors and a monoclonal antibody (HV-1) recognized epitopes in the amino terminus of the C2-domain (residues 2037-2087) and blocked PS binding. We have now investigated the function of individual amino acids within the C2-domain using charge to alanine mutagenesis. Charged residues located within the C2-domain were changed to alanine in clusters of 1-3 mutations per construct. In addition, mutants W2063A, W2064A, (W2063, W2064)A, and L2116A were constructed as well. The resultant 30 mutants were expressed in COS cells using a B-domain deleted factor V construct (rHFV des B). All mutants were expressed efficiently based on the polyclonal antibody ELISA. The charged residues, Arg(2074), Asp(2098), Arg(2171), Arg(2174), and Glu(2189) are required for maintaining the structural integrity of the C2-domain of factor V. Four of these residues (Arg(2074), Asp(2098), Arg(2171), and Arg(2174)) correspond to positions in the factor VIII C-type domains that have been identified as point mutations in patients with hemophilia A. The epitope for the inhibitory monoclonal antibody HV-1 has been localized to Lys(2060) through Glu(2069) in the factor V C2-domain. The epitope for the inhibitory monoclonal antibody 6A5 is composed of amino acids His(2128) through Lys(2137). The PS-binding site in the factor V C2-domain includes amino acid residues Trp(2063) and Trp(2064). This site overlaps with the epitope for monoclonal antibody HV-1. These factor V C2-domain mutants should provide valuable tools for further defining the molecular interactions responsible for factor V binding to phospholipid membranes.

Alanine↗

PubMed2000

The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium.

Calpains (calcium-dependent cytoplasmic cysteine proteinases) are implicated in processes such as cytoskeleton remodeling and signal transduction. The 2.3-A crystal structure of full-length heterodimeric [80-kDa (dI-dIV) + 30-kDa (dV+dVI)] human m-calpain crystallized in the absence of calcium reveals an oval disc-like shape, with the papain-like catalytic domain dII and the two calmodulin-like domains dIV+dVI occupying opposite poles, and the tumor necrosis factor alpha-like beta-sandwich domain dIII and the N-terminal segments dI+dV located between. Compared with papain, the two subdomains dIIa+dIIb of the catalytic unit are rotated against one another by 50 degrees, disrupting the active site and the substrate binding site, explaining the inactivity of calpains in the absence of calcium. Calcium binding to an extremely negatively charged loop of domain dIII (an electrostatic switch) could release the adjacent barrel-like subdomain dIIb to move toward the helical subdomain dIIa, allowing formation of a functional catalytic center. This switch loop could also mediate membrane binding, thereby explaining calpains' strongly reduced calcium requirements in vivo. The activity status at the catalytic center might be further modulated by calcium binding to the calmodulin domains via the N-terminal linkers.

Amino Acid Sequence↗

PubMed2000

Recognition and catabolism of synthetic heterotrimeric collagen peptides by matrix metalloproteinases.

BACKGROUND: The general consensus is that interstitial collagens are digested by collagenases and denatured collagen by gelatinases, although processing of fibrillar and acetic-acid-soluble collagen by gelatinase A has also been reported. One of the main difficulties in studying the mechanism of action of these matrix metalloproteinases (MMPs) derives from the physicochemical properties of the natural triple-helical collagen, which makes it difficult to handle. RESULTS: Synthetic heterotrimeric collagenous peptides that contain the collagenase cleavage site of human collagen type I and differ in the thermal stability of the triple-helical fold were used to mimic natural collagen and gelatin, respectively. Results from digestion of these substrates by fibroblast and neutrophil collagenases (MMP-1 and MMP-8), as well as by gelatinase A (MMP-2), confirmed that the two classes of enzymes operate within the context of strong conformational dependency of the substrates. It was also found that gelatinases and collagenases exhibit two distinct proteolytic mechanisms: gelatinase digests the gelatin-like heterotrimer rapidly in individual steps with intermediate releases of partially processed substrate into the medium, whereas collagenases degrade the triple-helical heterotrimer by trapping it until scission through all three alpha chains is achieved. CONCLUSIONS: The results confirm the usefulness of synthetic heterotrimeric collagenous peptides in the folded and unfolded state as mimics of the natural substrates collagen and gelatin, respectively, to gain a better a insight into the proteolytic mechanisms of matrix metalloproteinases.

Binding Sites↗

PubMed2000

Structure of a novel leech carboxypeptidase inhibitor determined free in solution and in complex with human carboxypeptidase A2.

Leech carboxypeptidase inhibitor (LCI) is a novel protein inhibitor present in the medicinal leech Hirudo medicinalis. The structures of LCI free and bound to carboxypeptidase A2 (CPA2)have been determined by NMR and X-ray crystallography, respectively. The LCI structure defines a new protein motif that comprises a five-stranded antiparallel beta-sheet and one short alpha-helix. This structure is preserved in the complex with human CPA2 in the X-ray structure, where the contact regions between the inhibitor and the protease are defined. The C-terminal tail of LCI becomes rigid upon binding the protease as shown in the NMR relaxation studies, and it interacts with the carboxypeptidase in a substrate-like manner. The homology between the C-terminal tails of LCI and the potato carboxypeptidase inhibitor represents a striking example of convergent evolution dictated by the target protease. These new structures are of biotechnological interest since they could elucidate the control mechanism of metallo-carboxypeptidases and could be used as lead compounds for the search of fibrinolytic drugs.

Amino Acid Sequence↗

PubMed2000

L-Isoaspartate 115 of porcine beta-trypsin promotes crystallization of its complex with bdellastasin.

Bdellastasin is a 59-amino-acid, cysteine-rich, antistasin-type inhibitor of sperm acrosin, plasmin and trypsin, isolated from the medicinal leech Hirudo medicinalis. The complex formed between bdellastasin and porcine beta-trypsin has previously been crystallized in the presence of PEG in a tetragonal crystal form of space group P4(3)2(1)2 and has now been found to crystallize under high-salt conditions in the enantiomorphic space group P4(1)2(1)2. These structures have been solved and refined to 2.8 and 2.7 A resolution, respectively. Bdellastasin turns out to have an antistasin-like fold exhibiting a bis-domainal structure. In the second new crystal form, the flexible N-terminal subdomain is rotated with respect to the C--terminal subdomain by about 90 degrees, fitting into a cavity formed by symmetry-related trypsin molecules. The canonical inhibitor-proteinase interaction is restricted to the primary binding loop comprising residues Leu31-Lys36 of bdellastasin. During the refinement, a bound sodium ion occupying the calcium-binding site of the porcine beta-trypsin component was discovered. This sodium ion is coordinated in a tetragonal-pyramidal manner, with the geometry of the enclosing loop slightly changed compared with the loop in the presence of calcium. In the crystal form of space group P4(3)2(1)2, the electron density for residue 115 of porcine beta-trypsin clearly indicates the presence of a beta-isomerized L-aspartic acid, which is placed in spatial proximity to segment Thr144--Gly148 of a symmetry-related trypsin molecule. This is the first structurally observed example of an L-isoaspartate in beta--trypsin originating from Asn. A comparison with other known crystal structures of porcine beta-trypsin-macromolecular inhibitor complexes suggests that the deamidation, isomerization and racemization of Asn115 is the key step in crystallization.

Animals↗

PubMed2000

Crystallization and preliminary X-ray analysis of recombinant full-length human m-calpain.

m-Calpain constitutes the prototype of the superfamily of neutral calcium-activated cysteine proteinases. It is a heterodimer consisting of an 80 and a 30 kDa subunit. Recombinant full-length human m-calpain has been crystallized using macro-seeding techniques and vapour-diffusion methods. Two different monoclinic crystal forms (space group P2(1)) were obtained from a solution containing polyethylene glycol (M(W) = 10 000) as a precipitating agent. Complete data sets have been collected to 2.3 and 3.0 A resolution using cryo-cooling conditions and synchrotron radiation. The unit-cell parameters are a = 64.86, b = 133.97, c = 78.00 A, beta = 102.43 degrees and a = 51.80, b = 171.36, c = 64.66 A, beta = 94.78 degrees, respectively. The V(m) values indicate that there is one heterodimer in each asymmetric unit.

Calpain↗

PubMed2000

Urethanyl-3-amidinophenylalanine derivatives as inhibitors of factor Xa. X-ray crystal structure of a trypsin/inhibitor complex and modeling studies.

Hydrophobic urethanyl derivatives of 3-amidinophenylalanine methyl ester were found to be relatively potent and selective factor Xa inhibitors. These compounds consist of the arginine-mimetic 3-benzamidino group as P1 residue and of hydrophobic residues as potential interaction partners for the S3/S4 aryl binding site of the enzyme. Attempts to possibly identify their binding mode to factor Xa via the X-ray crystal structure of a trypsin/inhibitor complex and analogy modeling on the crystal structure of factor Xa failed. However, synthesis of enantiomerically pure (R)- and (S)-derivatives, combined with modeling experiments, led to an hypothetical non-substrate like binding mode, which was fully confirmed by the remarkably enhanced inhibitory potency of derivatives in which the methyl ester was replaced by arylamides for interactions with the S3/S4 enzyme binding subsites. With adamantyloxycarbonyl-(R)-3-amidinophenylalanine-phenethylamide+ ++ a nanomolar inhibiton was obtained, thus indicating this new class of factor Xa inhibitors as a highly promising lead structure.

Animals↗

PubMed2000

Crystal structure of the caspase activator human granzyme B, a proteinase highly specific for an Asp-P1 residue.

Granzyme B is the prototypic member of the granzymes, a family of trypsin-like serine proteinases localized in the dense cytoplasmic granules of activated natural killer cells and cytotoxic T lymphocytes. Granzyme B directly triggers apoptosis in target cells by activating the caspase pathway, and has been implicated in the etiology of rheumatoid arthritis. Human granzyme B expressed in a baculovirus system has been crystallized without inhibitor and its structure has been determined to 3.1 A resolution, after considerably improving the diffraction power of the crystals by controlled humidity changes. The granzyme B structure reveals an overall fold similar to that found in cathepsin G and human chymase. The guanidinium group of Arg226, anchored at the back of the S1-specificity pocket, can form a salt bridge with the P1-Asp side chain of a bound peptide substrate. The architecture of the substrate binding site of granzyme B appears to be designed to accommodate and cleave hexapeptides such as the sequence Ile-Glu-Thr-Asp-/Ser-Gly present in the activation site of pro-caspase-3, a proven physiological substrate of granzyme B. These granzyme B crystals, with fully accessible active sites, are well suited for soaking with small synthetic inhibitors that might be used for a treatment of chronic inflammatory disorders.

Amino Acid Sequence↗

PubMed1999

Crystal structures of the membrane-binding C2 domain of human coagulation factor V.

Rapid and controlled clot formation is achieved through sequential activation of circulating serine proteinase precursors on phosphatidylserine-rich procoagulant membranes of activated platelets and endothelial cells. The homologous complexes Xase and prothrombinase, each consisting of an active proteinase and a non-enzymatic cofactor, perform critical steps within this coagulation cascade. The activated cofactors VIIIa and Va, highly specific for their cognate proteinases, are each derived from precursors with the same A1-A2-B-A3-C1-C2 architecture. Membrane binding is mediated by the C2 domains of both cofactors. Here we report two crystal structures of the C2 domain of human factor Va. The conserved beta-barrel framework provides a scaffold for three protruding loops, one of which adopts markedly different conformations in the two crystal forms. We propose a mechanism of calcium-independent, stereospecific binding of factors Va and VIIIa to phospholipid membranes, on the basis of (1) immersion of hydrophobic residues at the apices of these loops in the apolar membrane core; (2) specific interactions with phosphatidylserine head groups in the groove enclosed by these loops; and (3) favourable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups.

Amino Acid Sequence↗

PubMed1999

The variable region-1 from tissue-type plasminogen activator confers specificity for plasminogen activator inhibitor-1 to thrombin by facilitating catalysis: release of a kinetic block by a heterologous protein surface loop.

Substitution of the native variable region-1 (VR1/37-loop) of thrombin by the corresponding VR1 of tissue-type plasminogen activator (thrombin-VR1(tPA)) increases the rate of inhibition by plasminogen activator inhibitor type 1 (PAI-1) by three orders of magnitude, and is thus sufficient to confer PAI-1 specificity to a heterologous serine protease. A structural and kinetical approach to establish the function of the VR1 loop of t-PA in the context of the thrombin-VR1(tPA) variant is described. The crystal structure of thrombin-VR1(tPA) was resolved and showed a conserved overall alpha-thrombin structure, but a partially disordered VR1 loop as also reported for t-PA. The contribution of a prominent charge substitution close to the active site was studied using charge neutralization variants thrombin-E39Q(c39) and thrombin-VR1(tPA)-R304Q(c39), resulting in only fourfold changes in the PAI-1 inhibition rate. Surface plasmon resonance revealed that the affinity of initial reversible complex formation between PAI-1 and catalytically inactive Ser195-->Ala variants of thrombin and thrombin-VR1(tPA) is only increased fivefold, i.e. KD is 652 and 128 nM for thrombin-S195A and thrombin-S195A-VR1(tPA), respectively. We established that the partition ratio of the suicide substrate reaction between the proteases and PAI-1 was largely unaffected in any variant studied. Hirugen allosterically decreases the rate of thrombin inhibition by PAI-1 2.5-fold and of thrombin-VR1(tPA) 20-fold, by interfering with a unimolecular step in the reaction, not by decreasing initial complex formation or by altering the stoichiometry. Finally, kinetic modeling demonstrated that acylation is the rate-limiting step in thrombin inhibition by PAI-1 (k approximately 10(-3) s(-1)) and this kinetic block is alleviated by the introduction of the tPA-VR1 into thrombin (k>1 s(-1)). We propose that the length, flexibility and different charge architecture of the VR1 loop of t-PA invoke an induced fit of the reactive center loop of PAI-1, thereby enhancing the rate of acylation in the Michaelis complex between thrombin-VR1(t-PA) and PAI-1 by more than two orders of magnitude.

Acylation↗

PubMed1999

Structure of the complex of the antistasin-type inhibitor bdellastasin with trypsin and modelling of the bdellastasin-microplasmin system.

The serine proteinase plasmin is, together with tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), involved in the dissolution of blood clots in a fibrin-dependent manner. Moreover, plasmin plays a key role in a variety of other activation cascades such as the activation of metalloproteinases, and has also been implicated in wound healing, pathogen invasion, cancer invasion and metastasis. The leech-derived (Hirudo medicinalis) antistasin-type inhibitor bdellastasin represents a specific inhibitor of trypsin and plasmin and thus offers a unique opportunity to evaluate the concept of plasmin inhibition. The complexes formed between bdellastasin and bovine as well as porcine beta-trypsin have been crystallised in a monoclinic and a tetragonal crystal form, containing six molecules and one molecule per asymmetric unit, respectively. Both structures have been solved and refined to 3.3 A and 2.8 A resolution. Bdellastasin turns out to have an antistasin-like fold exhibiting a bis-domainal structure like the tissue kallikrein inhibitor hirustasin. The interaction between bdellastasin and trypsin is restricted to the C-terminal subdomain of bdellastasin, particularly to its primary binding loop, comprising residues Asp30-Glu38. The reactive site of bdellastasin differs from other antistasin-type inhibitors of trypsin-like proteinases, exhibiting a lysine residue instead of an arginine residue at P1. A model of the bdellastasin-microplasmin complex has been created based on the X-ray structures. Our modelling studies indicate that both trypsin and microplasmin recognise bdellastasin by interactions which are characteristic for canonically binding proteinase inhibitors. On the basis of our three-dimensional structures, and in comparison with the tissue-kallikrein-bound and free hirustasin and the antistasin structures, we postulate that the binding of the inhibitors toward trypsin and plasmin is accompanied by a switch of the primary binding loop segment P5-P3. Moreover, in the factor Xa inhibitor antistasin, the core of the molecule would prevent an equivalent rotation of the P3 residue, making exosite interactions of antistasin with factor Xa imperative. Furthermore, Arg32 of antistasin would clash with Arg175 of plasmin, thus impairing a favourable antistasin-plasmin interaction and explaining its specificity.

Amino Acid Sequence↗

PubMed1999

Crystal structure of gingipain R: an Arg-specific bacterial cysteine proteinase with a caspase-like fold.

Gingipains are cysteine proteinases acting as key virulence factors of the bacterium Porphyromonas gingivalis, the major pathogen in periodontal disease. The 1.5 and 2.0 A crystal structures of free and D-Phe-Phe-Arg-chloromethylketone-inhibited gingipain R reveal a 435-residue, single-polypeptide chain organized into a catalytic and an immunoglobulin-like domain. The catalytic domain is subdivided into two subdomains comprising four- and six-stranded beta-sheets sandwiched by alpha-helices. Each subdomain bears topological similarities to the p20-p10 heterodimer of caspase-1. The second subdomain harbours the Cys-His catalytic diad and a nearby Glu arranged around the S1 specificity pocket, which carries an Asp residue to enforce preference for Arg-P1 residues. This gingipain R structure is an excellent template for the rational design of drugs with a potential to cure and prevent periodontitis. Here we show the binding mode of an arginine-containing inhibitor in the active-site, thus identifying major interaction sites defining a suitable pharmacophor.

Adhesins, Bacterial↗