Biomedical subjects

W Bode

Publications and source records attributed to W Bode.

At least 19 recordsLinked to original sources

PubMed1999

The 2.2 A crystal structure of human chymase in complex with succinyl-Ala-Ala-Pro-Phe-chloromethylketone: structural explanation for its dipeptidyl carboxypeptidase specificity.

Human chymase (HC) is a chymotrypsin-like serine proteinase expressed by mast cells. The 2.2 A crystal structure of HC complexed to the peptidyl inhibitor, succinyl-Ala-Ala-Pro-Phe-chloromethylketone (CMK), was solved and refined to a crystallographic R-factor of 18.4 %. The HC structure exhibits the typical folding pattern of a chymotrypsin-like serine proteinase, and shows particularly similarity to rat chymase 2 (rat mast cell proteinase II) and human cathepsin G. The peptidyl-CMK inhibitor is covalently bound to the active-site residues Ser195 and His57; the peptidyl moiety juxtaposes the S1 entrance frame segment 214-217 by forming a short antiparallel beta-sheet. HC is a highly efficient angiotensin-converting enzyme. Modeling of the chymase-angiotensin I interaction guided by the geometry of the bound chloromethylketone inhibitor indicates that the extended substrate binding site contains features that may generate the dipeptidyl carboxypeptidase-like activity needed for efficient cleavage and activation of the hormone. The C-terminal carboxylate group of angiotensin I docked into the active-site cleft, with the last two residues extending beyond the active site, is perfectly localized to make a favorable hydrogen bond and salt bridge with the amide nitrogen of the Lys40-Phe41 peptide bond and with the epsilon-ammonium group of the Lys40 side-chain. This amide positioning is unique to the chymase-related proteinases, and only chymases from primates possess a Lys residue at position 40. Thus, the structure conveniently explains the preferred conversion of angiotensin I to angiotensin II by human chymase.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Structural and functional analyses of benzamidine-based inhibitors in complex with trypsin: implications for the inhibition of factor Xa, tPA, and urokinase.

The trypsin-like serine proteinase superfamily contains a number of potential therapeutic targets, many of which are unsuitable for routine X-ray crystallographic studies. We have cocrystallized a selection of benzamidine-based inhibitors with bovine trypsin and solved their structures to a resolution of up to 1.7 A. Despite similar chemical formulas, the inhibitors exhibit a range of diverse binding modes that reflect their inhibitory spectra against the serine proteinases trypsin, thrombin, factor Xa, tissue-type plasminogen activator (tPA) and urokinase (uPA). In contrast to the compact folded conformations of thrombin inhibitors which allow optimal binding in the well-defined hydrophobic S2/S4 pocket of thrombin, those effective against factor Xa exhibit an extended conformation that allows occupation of the S3/S4 region, where hydrophobic and electrostatic interactions can stabilize the conformation. One group of inhibitors containing an N-terminal 2,4, 6-triisopropylphenylsulfonyl (TIPPS) moiety show little or no penetration into the S3/S4 subsites of trypsin. These latter sites are occluded in uPA, explaining why this class of compounds is effective against uPA. Despite presenting an extensive hydrophobic surface toward the solvent, the Ki values for TIPPS-containing compounds against trypsin is in the range 10(-7) to 10(-8) M. Comparison of the binding of a bis-benzamidine inhibitor in trypsin and tPA indicate that a shift in potency can be induced by relatively minor changes in binding mode. Implications for the inhibition of these proteinases are discussed.

Animals↗

PubMed1998

Three-dimensional structure of human tissue inhibitor of metalloproteinases-2 at 2.1 A resolution.

The three-dimensional structure of human tissue inhibitor of metalloproteinases-2 (TIMP-2) was determined by X-ray crystallography to 2.1 A resolution. The structure of the inhibitor consists of two domains. The N-terminal domain (residues 1-110) is folded into a beta-barrel, similar to the oligonucleotide/oligosaccharide binding fold otherwise found in certain DNA-binding proteins. The C-terminal domain (residues 111-194) contains a parallel stranded beta-hairpin plus a beta-loop-beta motif. Comparison of the structure of uncomplexed human TIMP-2 with that of bovine TIMP-2 bound to the catalytic domain of human MMP-14 suggests an internal rotation between the two domains of approximately 13 degrees upon binding to the protease. Furthermore, local conformational differences in the two structures that might be induced by formation of the protease-inhibitor complex have been found. The most prominent of these involves residues 27-40 of the A-B beta-hairpin loop. Structure-based alignment of amino acid sequences of representatives of the TIMP family maps the sequence differences mainly to loop regions, and some of these differences are proposed to be responsible for the particular properties of the various TIMP species.

Amino Acid Sequence↗

PubMed1998

Design of benzamidine-type inhibitors of factor Xa.

A series of derivatives of rac-benzenesulfonyl-glycyl-phenylalanine or its ethyl ester with a combination of thioamido/amidino or amidino/amidino substituents in the benzene rings was synthesized as potential inhibitors of factor Xa (fXa). Among these, the racemic 4'-amidinobenzenesulfonyl-glycyl-4-amidinophenylalanine ethyl ester was found to exhibit the highest affinity for fXa despite the unfavored location of the amidino substituent in the para position. X-ray structural analysis of the trypsin complex with this bis-benzamidine compound revealed a retro-binding mode if compared to those of similar compounds, so far analyzed in complexes with trypsin or fXa. This noncanonical binding mode as well as its slow plasma clearance rates in rats, if compared to those of other benzamidine derivatives, suggests this compound as an interesting new lead structure for the design of fXa inhibitors.

Animals↗

PubMed1998

Amino-acid sequence and three-dimensional structure of the Staphylococcus aureus metalloproteinase at 1.72 A resolution.

BACKGROUND: Aureolysin is an extracellular zinc-dependent metalloproteinase from the pathogenic bacterium Staphylococcus aureus. This enzyme exhibits in vitro activity against several molecules of biological significance for the host, indicating that it is involved in the pathology of staphylococcal diseases. RESULTS: Here we report the amino-acid sequence and inhibitor-free X-ray crystal structure of aureolysin, a member of the thermolysin family of zinc-dependent metalloproteinases. This enzyme, which binds one zinc and three calcium ions, comprises a single chain of 301 amino acids that consists of a beta-strand-rich upper domain and an alpha-helix-rich lower domain. CONCLUSIONS: The overall structure of aureolysin is very similar to that of the other three members of this family whose structures are known - thermolysin (TLN) from Bacillus thermoproteolyticus, neutral protease (NP) from Bacillus cereus and elastase (PAE) from Pseudomonas aeruginosa. But an important difference has been encountered: in contrast to what has been observed in the other three members of this family (TLN, NP and PAE), inhibitor-free aureolysin displays a 'closed' active site cleft conformation. This new structure therefore raises questions about the universality of the hinge-bending motion model for the neutral metalloproteinases.

Amino Acid Sequence↗

PubMed1998

The crystal structure of the novel snake venom plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases.

BACKGROUND: Trimeresurus stejnejeri venom plasminogen activator (TSV-PA) is a snake venom serine proteinase that specifically activates plasminogen. Snake venom serine proteinases form a subfamily of trypsin-like proteinases that are characterised by a high substrate specificity and resistance to inhibition. Many of these venom enzymes specifically interfere with haemostatic mechanisms and display a long circulating half-life. For these reasons several of them have commercial applications and are potentially attractive pharmacological tools. RESULTS: The crystal structure of TSV-PA has been determined to 2.5 A resolution and refined to an R factor of 17.8 (R free, 24.4). The enzyme, showing the overall polypeptide fold of trypsin-like serine proteinases, displays unique structural elements such as the presence of a phenylalanine at position 193, a C-terminal tail clamped via a disulphide bridge to the 99-loop, and a structurally conserved Asp97 residue. The presence of a cis proline at position 218 is in agreement with evolutionary relationships to glandular kallikrein. CONCLUSIONS: We postulate that Phe 193 accounts for the high substrate specificity of TSV-PA and renders it incapable of forming a stable complex with bovine pancreatic trypsin inhibitor and other extended substrates and inhibitors. Mutational studies previously showed that Asp97 is crucial for the plasminogenolytic activity of TSV-PA, here we identify the conservation of Asp97 in both types of mammalian plasminogen activator - tissue-type (tPA) and urokinase-type (uPA). It seems likely that Asp97 of tPA and uPA will have a similar role in plasminogen recognition. The C-terminal extension of TSV-PA is conserved among snake venom serine proteinases, although its function is unknown. The three-dimensional structure presented here is the first of a snake venom serine proteinase and provides an excellent template for modelling other homologous family members.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor.

The proteolytic activity of matrix metalloproteinases (MMPs) towards extracellular matrix components is held in check by the tissue inhibitors of metalloproteinases (TIMPs). The binary complex of TIMP-2 and membrane-type-1 MMP (MT1-MMP) forms a cell surface located 'receptor' involved in pro-MMP-2 activation. We have solved the 2.75 A crystal structure of the complex between the catalytic domain of human MT1-MMP (cdMT1-MMP) and bovine TIMP-2. In comparison with our previously determined MMP-3-TIMP-1 complex, both proteins are considerably tilted to one another and show new features. CdMT1-MMP, apart from exhibiting the classical MMP fold, displays two large insertions remote from the active-site cleft that might be important for interaction with macromolecular substrates. The TIMP-2 polypeptide chain, as in TIMP-1, folds into a continuous wedge; the A-B edge loop is much more elongated and tilted, however, wrapping around the S-loop and the beta-sheet rim of the MT1-MMP. In addition, both C-terminal edge loops make more interactions with the target enzyme. The C-terminal acidic tail of TIMP-2 is disordered but might adopt a defined structure upon binding to pro-MMP-2; the Ser2 side-chain of TIMP-2 extends into the voluminous S1' specificity pocket of cdMT1-MMP, with its Ogamma pointing towards the carboxylate of the catalytic Glu240. The lower affinity of TIMP-1 for MT1-MMP compared with TIMP-2 might be explained by a reduced number of favourable interactions.

Amino Acid Sequence↗

PubMed1998

New enzyme lineages by subdomain shuffling.

Protein functions have evolved in part via domain recombination events. Such events, for example, recombine structurally independent functional domains and shuffle targeting, regulatory, and/or catalytic functions. Domain recombination, however, can generate new functions, as implied by the observation of catalytic sites at interfaces of distinct folding domains. If useful to an evolving organism, such initially rudimentary functions would likely acquire greater efficiency and diversity, whereas the initially distinct folding domains would likely develop into single functional domains. This represents the probable evolution of the S1 serine protease family, whose two homologous beta-barrel subdomains assemble to form the binding sites and the catalytic machinery. Among S1 family members, the contact interface and catalytic residues are highly conserved whereas surrounding surfaces are highly variable. This observation suggests a new strategy to engineer viable proteins with novel properties, by swapping folding subdomains chosen from among protein family members. Such hybrid proteins would retain properties conserved throughout the family, including folding stability as single domain proteins, while providing new surfaces amenable to directed evolution or engineering of specific new properties. We show here that recombining the N-terminal subdomain from coagulation factor X with the C-terminal subdomain from trypsin creates a potent enzyme (fXYa) with novel properties, in particular a broad substrate specificity. As shown by the 2.15-A crystal structure, plasticity at the hydrophobic subdomain interface maintains activity, while surface loops are displaced compared with the parent subdomains. fXYa thus represents a new serine proteinase lineage with hybrid fX, trypsin, and novel properties.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Bis-substituted malonic acid hydroxamate derivatives as inhibitors of human neutrophil collagenase (MMP8).

Malonic acid hydroxamate derivatives bis-substituted at the methylene group were synthesized as potential nonpeptidic inhibitors of human neutrophil collagenase (MMP8). The presence of an aromatic residue both at the C2 malonic acid position and in the C-terminal tail for hydrophobic interactions with the surface-exposed S1 binding site and the S1' pocket of the enzyme, respectively, was found to be sufficient for submicromolar inhibition potencies. For optimal insertion of the aryl amide group into the hydrophobic S1' pocket, spacing of the C-terminal phenyl group by at least a 3C-chain was required. In view of these results the achiral indan-2, 2-dicarboxylic acid was used to mimic the 2-benzyl-2-methylmalonic acid residue, and its derivatization to the 3-phenylpropyl amide hydroxamate produced a potent, achiral, low-mass inhibitor of MMP8 (Ki = 0.3 microM), the binding mode of which was unambiguously determined by X-ray crystallographic analysis.

Binding Sites↗

PubMed1998

Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity.

This study was designed to identify functionally important factor IX (FIX) residues. Using recombinant techniques and cell culture, we produced a mutant FIX with arginine at 338 changed to alanine (R338A-FIX). This molecule had approximately 3 times greater clotting activity than that of wild type FIX (wt-FIX) in the activated partial thromboplastin assay. R338A-FIX reacted normally with a panel of three FIX specific monoclonal antibodies and migrated on sodium dodecyl sulfate-polyacrylamide gels indistinguishably from wt-FIX. Using functional assays, we determined that R338A-FIXa's Kd for factor VIIIa (FVIIIa) was similar to that of wt-FIXa. Our kinetic analysis, using factor X as substrate, indicated that the mutation's major effects were a 3-fold increase in kcat and a 2-fold decrease in Km both manifested only in the presence of FVIIIa. R338A-FIXa's increased catalytic efficiency did not result from ablation of a thrombin sensitive site, reported to occur at arginine 338, since in our assays the thrombin inhibitor, hirudin, had no effect on activity of either wt-FIXa or R338A-FIXa. R338A-FIXa and wt-FIXa had equal activity, with or without FVIIIa, toward the synthetic substrate, methylsulfonyl-D-cyclohexylglycyl-arginine-p-nitroanilide. Interestingly, R338A-FIXa had reduced affinity for heparin. Therefore, we propose that R338A-FIXa's increased activity is not due to an allosteric effect on the active site, but that the Arg-338 residue is part of an exosite that binds both factor X and the mucopolysaccharide, heparin.

Alanine↗

PubMed1998

Crystal structure of the catalytic domain of human tumor necrosis factor-alpha-converting enzyme.

Tumor necrosis factor-alpha (TNFalpha) is a cytokine that induces protective inflammatory reactions and kills tumor cells but also causes severe damage when produced in excess, as in rheumatoid arthritis and septic shock. Soluble TNFalpha is released from its membrane-bound precursor by a membrane-anchored proteinase, recently identified as a multidomain metalloproteinase called TNFalpha-converting enzyme or TACE. We have cocrystallized the catalytic domain of TACE with a hydroxamic acid inhibitor and have solved its 2.0 A crystal structure. This structure reveals a polypeptide fold and a catalytic zinc environment resembling that of the snake venom metalloproteinases, identifying TACE as a member of the adamalysin/ADAM family. However, a number of large insertion loops generate unique surface features. The pro-TNFalpha cleavage site fits to the active site of TACE but seems also to be determined by its position relative to the base of the compact trimeric TNFalpha cone. The active-site cleft of TACE shares properties with the matrix metalloproteinases but exhibits unique features such as a deep S3' pocket merging with the S1' specificity pocket below the surface. The structure thus opens a different approach toward the design of specific synthetic TACE inhibitors, which could act as effective therapeutic agents in vivo to modulate TNFalpha-induced pathophysiological effects, and might also help to control related shedding processes.

ADAM Proteins↗

PubMed1998

Human beta-tryptase is a ring-like tetramer with active sites facing a central pore.

Human tryptase, a mast-cell-specific serine proteinase that may be involved in causing asthma and other allergic and inflammatory disorders, is unique in two respects: it is enzymatically active only as a heparin-stabilized tetramer, and it is resistant to all known endogenous proteinase inhibitors. The 3-A crystal structure of human beta-tryptase in a complex with 4-amidinophenyl pyruvic acid shows four quasi-equivalent monomers arranged in a square flat ring of pseudo 222 symmetry. Each monomer contacts its neighbours at two different interfaces through six loop segments. These loops are located around the active site of beta-tryptase and differ considerably in length and conformation from loops of other trypsin-like proteinases. The four active centres of the tetramer are directed towards an oval central pore, restricting access for macromolecular substrates and enzyme inhibitors. Heparin chains might stabilize the complex by binding to an elongated patch of positively charged residues spanning two adjacent monomers. The nature of this unique tetrameric architecture explains many of tryptase's biochemical properties and provides a basis for the rational design of monofunctional and bifunctional tryptase inhibitors.

Amino Acid Sequence↗

PubMed1998

Cutting at the right place--the importance of selective limited proteolysis in the activation of proproteinase E.

Proteinase E is a proteolytic enzyme which belongs to a distinct subfamily of chymotrypsin-like serine endopeptidases. Its proform from the bovine pancreatic system has been structurally analyzed by X-ray crystallography for the intact native form, with a 11-residue N-terminal activation peptide, in a ternary complex with chymotrypsinogen C and procarboxypeptidase A [Gomis-Rüth, F. X., Gómez, M., Bode, W., Huber, R. & Avilés, F. X. (1995) The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C, EMBO J. 14, 4387-4394]. Also for a N-terminally truncated form, lacking the first 13 residues and called subunit III, a crystal structure is available [Pignol, D., Gaboriaud, C., Michon, T., Kerfelec, B., Chapus, C. & Fontecilla-Camps, J. C. (1994) Crystal structure of bovine procarboxypeptidase A-S6 subunit III, a highly structured truncated zymogen E, EMBO J. 8, 1763-1771]. Both structures are well defined by electron density, except for the first 7 residues of subunit III. However, both structures present large deviations of up to 2 nm in several regions, indicating that they correspond to two quite distinct states of low free energy, influenced by very few contacts made via the N-terminal segment. As no structure of an active proteinase E is known so far, pancreatic porcine elastase has been chosen as a model for this enzyme and an activation mechanism for this distinct serine endopeptidase subfamily is proposed.

Amino Acid Sequence↗

PubMed1998

Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family.

The proline iminopeptidase from Xanthomonas campestris pv. citri is a serine peptidase that catalyses the removal of N-terminal proline residues from peptides with high specificity. We have solved its three-dimensional structure by multiple isomorphous replacement and refined it to a crystallographic R-factor of 19.2% using X-ray data to 2.7 A resolution. The protein is folded into two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet flanked by two helices and the 11 N-terminal residues on one side, and by four helices on the other side. The smaller domain is placed on top of the larger domain and essentially consists of six helices. The active site, located at the end of a deep pocket at the interface between both domains, includes a catalytic triad of Ser110, Asp266 and His294. Cys269, located at the bottom of the active site very close to the catalytic triad, presumably accounts for the inhibition by thiol-specific reagents. The overall topology of this iminopeptidase is very similar to that of yeast serine carboxypeptidase. The striking secondary structure similarity to human lymphocytic prolyl oligopeptidase and dipeptidyl peptidase IV makes this proline iminopeptidase structure a suitable model for the three-dimensional structure of other peptidases of this family.

Amino Acid Sequence↗

PubMed1998

Crystallization and preliminary X-ray diffraction analysis of gingipain R2 from Porphyromonas gingivalis in complex with H-D-Phe-Phe-Arg-chloromethylketone.

Gingipain R2 is a 50 kDa proteinase from the oral pathogenic bacterium Porphyromonas gingivalis. This proteinase, which displays no significant sequence homology to any protein previously analyzed by X-ray crystallography, has been crystallized using the vapor diffusion method. Two different crystal forms were obtained from a solution containing polyethylene glycol (MW 8,000) (space group P2(1)2(1)2(1)) or magnesium sulfate (space group R3) as precipitating agent. Complete diffraction data sets have been collected up to 2.0 and 2.9 A resolution, respectively. Cell dimensions are a = 51.9 A, b = 79.9 A, and c = 99.6 A (P2(1)2(1)2(1)), and a = b = 176.6 A, and c = 143.4 A (R3). Considerations of the possible values of Vm accounts for the presence of one monomer per asymmetric unit in the case of the orthorhombic crystal form, whereas the rhombohedral crystal form, together with the analysis of the self-rotation function, could accommodate a tetramer in the asymmetric unit.

Adhesins, Bacterial↗

PubMed1998

Structure of malonic acid-based inhibitors bound to human neutrophil collagenase. A new binding mode explains apparently anomalous data.

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases, which have been implicated in various disease processes. Various classes of MMP inhibitors, including hydroxamic acids, phosphinic acids, and thiols, have been previously described. Most of these mimic peptides, and most likely bind analogous to the corresponding peptide substrates. Among the hydroxamic acids, malonic acid derivatives have been used as MMP inhibitors, although optimization of their inhibition potency was not successful. Here we report the design of malonic acid-based inhibitors using the X-ray structure of a collagenase/inhibitor complex, which revealed a nonsubstrate-like binding mode. The proposed beta-type turn-like conformation for the improved inhibitors was confirmed by X-ray crystallography. The observation of nonsubstrate-like binding confirms the original strategy for structure-based modeling of improved malonic acid inhibitors, and explains kinetic data that are inconsistent with substrate-like binding. Detailed interactions for the improved inhibitors seen in the crystal structure also suggest possibilities for further modifications in cycles of structure based drug design. Indeed, we have designed nonpeptidic inhibitors with approximately 500-fold improved inhibition based on these structures.

Collagenases↗

PubMed1998

The ternary microplasmin-staphylokinase-microplasmin complex is a proteinase-cofactor-substrate complex in action.

The serine proteinase plasmin is the key fibrinolytic enzyme that dissolves blood clots and also promotes cell migration and tissue remodeling. Here, we report the 2.65 A crystal structure of a ternary complex of microplasmin-staphylokinase bound to a second microplasmin. The staphylokinase 'cofactor' does not affect the active-site geometry of the plasmin 'enzyme', but instead modifies its subsite specificity by providing additional docking sites for enhanced presentation of the plasminogen 'substrate' to the 'enzymes's' active site. The activation loop of the plasmin 'substrate', cleaved in these crystals, can be reconstructed to show how it runs across the active site of the plasmin 'enzyme' prior to activation cleavage. This is the first experimental structure of a productive proteinase-cofactor-macromolecular substrate complex. Furthermore, it provides a template for the design of improved plasminogen activators and plasmin inhibitors with considerable therapeutical potential.

Amino Acid Sequence↗

PubMed1998

An active zymogen: unravelling the mystery of tissue-type plasminogen activator.

In contrast to almost all other proteinases, human tissue-type plasminogen activator (tPA) is also proteolytically active in its zymogen or single-chain form. The closely related plasminogen activator isolated from vampire bat saliva (vPA) acts exclusively in the single-chain form, lacking the requisite cleavage site for proteolytic activation. Recent structural studies on the proteolytic domains of vPA and human tPA in two- and single-chain forms reveal the mechanism of this anomalous activity. The PA-catalyzed proteolytic conversion of plasminogen to plasmin, responsible for the initiation of fibrinolysis, is fibrin-dependent; comparative structural analysis of the plasminogen activators provides clues as to the role of fibrin as cofactor.

Amino Acid Sequence↗