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W Bode

Publications and source records attributed to W Bode.

At least 73 records · Page 4Linked to original sources
PubMed1997

Crystal structure of an oligomer of proteolytic zymogens: detailed conformational analysis of the bovine ternary complex and implications for their activation.

The pancreas of ruminants secretes a 100 kDa non-covalent ternary complex of the zymogen of a metalloexopeptidase, carboxypeptidase A, and the proforms of two serine endopeptidases, chymotrypsin C and proteinase E. The crystal structure of the bovine complex has been solved and refined to an R-factor of 0.192 using synchrotron radiation X-ray data to 2.35 A resolution. In this heterotrimeric complex, the 403 residue procarboxypeptidase A takes a central position, with chymotrypsinogen C and proproteinase E attached to different surface sites of it. The procarboxypeptidase A subunit is composed of the active enzyme part and the 94 residue prodomain, similar to the monomeric porcine homologous form. The 251 residue subunit chymotrypsinogen structure, the first solved of an anionic (acidic pI) chymotrypsinogen, exhibits characteristics of both chymotrypsinogen A and elastases, with a potential specificity pocket of intermediate size (to accommodate apolar medium-sized residues) although not properly folded, as in bovine chymotrypsinogen A; this pocket displays a "zymogen triad" characteristic for zymogens of the chymotrypsinogen family, consisting of three non-catalytic residues (one serine, one histidine, and one aspartate) arranged in a fashion similar to the catalytic residues in the active enzymes. Following the traits of this family, the N terminus is clamped to the main molecular body by a disulphide bond, but the close six residue activation segment is completely disordered. The third zymogen, the 253 residue proproteinase E, bears close conformational resemblance to active porcine pancreatic elastase; its specificity pocket is buried, displaying the second "zymogen triad". Its five N-terminal residues are disordered, although the close activation site is fixed to the molecular surface. The structure of this native zymogen displays large conformational differences when compared with the recently solved crystal structure of bovine subunit III, an N-terminally truncated, non-activatable, proproteinase E variant lacking the first 13 residues of the native proenzyme. Most of the prosegment of procarboxypeptidase A and its activation sites are buried in the centre of the oligomer, whilst the activation sites of chymotrypsinogen C and proproteinase E are surface-located and not involved in intra or inter-trimer contacts. This organization confers a functional role to the oligomeric structure, establishing a sequential proteolytic activation for the different zymogens of the complex. The large surface and number of residues involved in the contacts among subunits, as well as the variety of non-bonded interactions, account for the high stability of the native ternary complex.

Animals↗
PubMed1997

The second Kunitz domain of human tissue factor pathway inhibitor: cloning, structure determination and interaction with factor Xa.

Tissue Factor Pathway Inhibitor (TFPI) is a 36 kDa glycoprotein that helps maintain haemostasis by inhibiting Factor Xa and the Factor VIIa/Tissue Factor (TF) complex. TFPI contains three tandemly linked Kunitz inhibitor domains, of which the second inhibits factor Xa. We have undertaken a multidisciplinary approach to study the structure and function of the second Kunitz domain of TFPI, with a view towards the rational design of factor Xa inhibitors. Amino acid residues 93 to 154 of the mature TFPI protein, corresponding to the second Kunitz domain (TFPI-kII), were expressed in Escherichia coli. The protein was purified to near homogeneity by ion exchange, hydrophobic interaction, and size exclusion chromatography, respectively. TFPI-kII is a potent factor Xa inhibitor with a Ki of 1.5 x 10(-10) M, a value that does not differ significantly from that of intact TFPI. The three-dimensional structure of TFPI-kII in aqueous solution was determined by 1H nuclear magnetic resonance spectroscopy (NMR). A set of 30 conformers was calculated with the program DIANA using 906 distance constraints derived from nuclear Overhauser effects and 23 dihedral angle constraints. This set, representing the solution structure of TFPI-kII, has an average root-mean-square deviation of 0.78 A for the backbone atoms and 1.38 A for all heavy atoms of residues 1 to 58. The structure of TFPI-kII has also been determined in complex with porcine trypsin using X-ray crystallographic techniques. The complex has been solved to a resolution of 2.6 A, with a final R-factor of 16.2%. Comparison of the NMR derived structure with that of TFPI-kII in complex with trypsin reveals little divergence of the two structures, with the exception of residue Tyr17. Superposition of the trypsin:TFPI-kII complex on factor Xa provides insights into macromolecular determinants for the inhibition of factor Xa. Complexation would require a degree of reorganisation of factor Xa residues, in particular of TyrF99, but also perhaps of the F148-loop. The interaction was further investigated using restrained molecular dynamics. Electrostatic interactions would appear to play a major role. The reorganisation of factor Xa is in contrast to the proposed factor Xa:TAP interaction, where TAP would bind to the "ground state" structure of factor Xa.

Amino Acid Sequence↗
PubMed1997

The thrombin E192Q-BPTI complex reveals gross structural rearrangements: implications for the interaction with antithrombin and thrombomodulin.

Previous crystal structures of thrombin indicate that the 60-insertion loop is a rigid moiety that partially occludes the active site, suggesting that this structural feature plays a decisive role in restricting thrombin's specificity. This restricted specificity is typified by the experimental observation that thrombin is not inhibited by micromolar concentrations of basic pancreatic trypsin inhibitor (BPTI). Surprisingly, a single atom mutation in thrombin (E192Q) results in a 10(-8) M affinity for BPTI. The crystal structure of human thrombin mutant E192Q has been solved in complex with BPTI at 2.3 A resolution. Binding of the Kunitz inhibitor is accompanied by gross structural rearrangements in thrombin. In particular, thrombin's 60-loop is found in a significantly different conformation. Concomitant reorganization of other surface loops that surround the active site, i.e. the 37-loop, the 148-loop and the 99-loop, is observed. Thrombin can therefore undergo major structural reorganization upon strong ligand binding. Implications for the interaction of thrombin with antithrombin and thrombomodulin are discussed.

Antithrombin III↗
PubMed1997

Identification of a hydrophobic exosite on tissue type plasminogen activator that modulates specificity for plasminogen.

A wide variety of important biological processes, including both the formation and dissolution of blood clots, depend on specific cleavage of individual target proteins by serine proteases. For example, tissue type plasminogen activator (t-PA), a trypsin-like enzyme that catalyzes the rate-limiting step of the endogenous fibrinolytic cascade, has only one known substrate in vivo, a single peptide bond (Arg561-Val562) in the proenzyme plasminogen. We have previously suggested that the specificity of t-PA for plasminogen is mediated in part by direct protein-protein interactions between the protease domain of t-PA and plasminogen that are distinct from those occurring within t-PA's active site. We demonstrate in this study that residues 420-423 of t-PA, which form a fully solvent-exposed, hydrophobic region of a surface loop mapping near one edge of the active site of t-PA, form, or are essential for the integrity of, an important, secondary site of interaction between t-PA and plasminogen that significantly modulates the rate of plasminogen activation in the absence, but not the presence, of fibrin. Identification of this secondary site of interaction between t-PA and plasminogen provides new insight into molecular details of the evolution of stringent substrate specificity by t-PA and suggests a novel strategy to enhance the fibrin dependence of plasminogen activation by t-PA. While the activity of wild type t-PA is stimulated by fibrin by a factor of approximately 650, the activity of two variants characterized in this study, t-PA/R275E,P422G and t-PA/R275E,P422E, is stimulated by a factor of approximately 39,000 or 61,000, respectively. It is therefore possible that, compared with wild type t-PA, the two variants would display enhanced "clot selectivity" in vivo due to reduced activity in the circulation but full activity at a site of fibrin deposition.

Animals↗
PubMed1997

Crystallization and preliminary X-ray diffraction analysis of proline iminopeptidase from Xanthomonas campestris pv. citri.

Proline iminopeptidase from Xanthomonas campestris pv. citri, displaying no significant sequence homology to any protein previously analyzed by X-ray crystallography, has been crystallized using the vapour diffusion method. Two different orthorhombic crystal forms (space group C222 and I222) were obtained from a solution containing NaCl or polyethylene glycol monomethyl ether (MW 5000) as precipitating agent for the native and lanthanum-derivatized protein, respectively. Complete diffraction data sets have been collected up to 2.6 A (native) and 3.0 A (lanthanum derivative) resolution. Cell dimensions are a= 147.2 A, b = 167.8 A, and c = 85.6 A (C222) and a = 146.7 A, b = 167.7 A, and c = 171.4 A (I222), respectively. Considerations of the possible values of V(m) and analysis of the self-rotation function of the native crystals account for the presence of one dimer per asymmetric unit, whereas a tetramer probably would occupy the smallest crystallographically independent crystal portion in the lanthanum-derivatized protein crystals.

Aminopeptidases↗
PubMed1997

Tissue-type plasminogen activator: variants and crystal/solution structures demarcate structural determinants of function.

NMR and crystal structure of many components of tissue-type plasminogen activator (t-PA) are now available: the finger-EGF pair and the kringle-2 domain structures have been solved, as have the proteolytic domains of vampire bat PA and human t-PA in two- and single-chain forms. These structures confirm the trypsin-like arrangement of the proteolytic domain of t-PA and show how surface loops near the catalytic centre contribute to the narrow specificity of t-PA. Together with mutational experiments, they identify the Lys156 sidechain as a cause of the amidolytic activity of single-chain t-PA, as it can provide a substitute salt bridge partner for Asp194 in the absence of the Ile16 N terminus of the two-chain form. These new findings provide new ideas for the design of PA variants with improved therapeutic properties.

Amino Acid Sequence↗
PubMed1997

Expression, purification, characterization, and X-ray analysis of selenomethionine 215 variant of leukocyte collagenase.

Matrix metalloproteinases belong to the superfamily of metzincins containing, besides a similar topology and a strictly conserved zinc environment, a 1,4-tight turn with a strictly conserved methionine residue at position three (the so called Met-turn [Bode et. al. (1993) FEBS 331, 134-140; Stöcker et al. (1995) Protein Sci. 4, 823-840]. The distal S-CH3 moiety of this methionine residue forms the hydrophobic basement of the three His residues liganding the catalytic zinc ion. To assess the importance of this methionine, we have expressed the catalytic domain of neutrophil collagenase (rHNC, residues Met80-Gly242) in the methionine auxotrophic Escherichia coli strain B834[DE3](hsd metB), with the two methionine residues replaced by selenomethionine. Complete replacement was confirmed by amino acid analysis and electrospray mass spectrometry. The folded and purified enzyme retained its catalytic activity, but showed modifications which are reflected in change kinetic parameters. The Met215SeMet substitution caused a decrease in conformational stability upon area denaturation. The X-ray crystal structure of this selenomethionine rHNC was virtually identical to that of the wild-type catalytic domain except for a very faint local disturbance around the sulfur-seleno substitution site.

Cloning, Molecular↗
PubMed1997

Characterization and application of a strain-specific monoclonal antibody against the rhizosphere bacterium Azospirillum brasilense Wa5.

A hybridoma cell line producing a rat monoclonal antibody (MAb Bo-33) directed against lipopolysaccharide of Azospirillum brasilense Wa5 has been established and characterized. Whole bacteria were used as immunogens. The number of antigens per cell was about 1500. The number of antigens per cell of reisolates from the rhizosphere of what was similar to the number of antigens of bacteria cultivated in rich medium. The sensitivity of detection using MAb Bo-33 was about 100 bacteria/ml. Therefore, the MAb was suitable for in situ immunofluorescence detection and a sensitive direct quantification of Azospirillum brasilense Wa5 in rhizosphere extracts.

Animals↗
PubMed1996

Crystal structure of a coagulogen, the clotting protein from horseshoe crab: a structural homologue of nerve growth factor.

The clotting cascade system of the horseshoe crab (Limulus) is involved in both haemostasis and host defence. The cascade results in the conversion of coagulogen, a soluble protein, into an insoluble coagulin gel. The clotting enzyme excises the fragment peptide C from coagulogen, giving rise to aggregation of the monomers. The crystal structure of coagulogen reveals an elongated molecule that embraces the helical peptide C fragment. Cleavage and removal of the peptide C would expose an extended hydrophobic cove, which could interact with the hydrophobic edge of a second molecule, leading to a polymeric fibre. The C-terminal half of the coagulogen molecule exhibits a striking topological similarity to the neurotrophin nerve growth factor (NGF), providing the first evidence for a neurotrophin fold in invertebrates. Similarities between coagulogen and Spatzle, the Drosophila ligand of the receptor Toll, suggest that the neurotrophin fold might be considered more ancient and widespread than previously realized.

Amino Acid Sequence↗
PubMed1996

The 2.8 A crystal structure of Gla-domainless activated protein C.

The structure of the Gla-domainless form of the human anticoagulant enzyme activated protein C has been solved at 2.8 A resolution. The light chain is composed of two domains: an epidermal growth factor (EGF)-like domain modified by a large insert containing an additional disulfide, followed by a typical EGF-like domain. The arrangement of the long axis of these domains describes an angle of approximately 80 degrees. Disulfide linked to the light chain is the catalytic domain, which is generally trypsin-like but contains a large insertion loop at the edge of the active site, a third helical segment, a prominent cationic patch analogous to the anion binding exosite I of thrombin and a trypsin-like Ca[II] binding site. The arrangement of loops around the active site partially restricts access to the cleft. The S2 and S4 subsites are much more polar than in factor Xa and thrombin, and the S2 site is unrestricted. While quite open and exposed, the active site contains a prominent groove, the surface of which is very polar with evidence for binding sites on the primed side, in addition to those typical of the trypsin class found on the non-primed side.

Binding Sites↗
PubMed1996

The helping hand of collagenase-3 (MMP-13): 2.7 A crystal structure of its C-terminal haemopexin-like domain.

Collagenase-3 (MMP-13) is a matrix metalloproteinase involved in human breast cancer pathology and in arthritic processes. The crystal structure of its C-terminal haemopexin-like domain has been solved by molecular replacement and refined to an R-value of 0.195 using data to 2.7 A resolution. This structure reveals a disk-like shape. The chain is folded into a beta-propeller structure of pseudo 4-fold symmetry, with the four propeller blades arranged around a funnel-like tunnel. This central tunnel tube harbours four ions assigned as two calcium and two chloride ions. The C-terminal domain of collagenase-3 has a similar structure to the equivalent domain of gelatinase A and fibroblast collagenase 1; however, its detailed structure and surface charge pattern has a somewhat greater similarity to the latter, in agreement with the subgrouping of MMP-13 with the collagenase subfamily of MMPs. It is proposed that several small structural differences may act together to confer the characteristic binding and cleavage specificities of collagenases for triple-helical substrates, probably in co-operation with a fitting interdomain linker.

Amino Acid Sequence↗
PubMed1996

Design and synthesis of heterotrimeric collagen peptides with a built-in cystine-knot. Models for collagen catabolism by matrix-metalloproteases.

A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site (P4-P'9/10) of type I collagen linked to a C-terminal cystine-knot, and N-terminally extended with (Gly-Pro-Hyp)5 triplets for stabilization of the triple-helical conformation. By employing a newly developed regioselective cysteine pairing strategy based exclusively on thiol disulfide exchange reactions, we succeeded in assembling in high yields and in a reproducible manner the triple-stranded cystine peptide. While the single chains showed no tendency to self-association into triple helices, the heterotrimer (alpha1 alpha2 alpha1') was found to exhibit a typical collagen-like CD spectrum at room temperature and a melting temperature (Tm) of 33 degrees C. This triple-helical collagen-like peptide is cleaved by the full-length human neutrophil collagenase (MMP-8) at a single locus fully confirming the correct raster of the heterotrimer. Its digestion proceeds at rates markedly higher than that of a single alpha1' chain. In contrast, opposite digestion rates were measured with the catalytic Phe79-MMP-8 domain of HNC. Moreover, the full-length enzyme exhibits Km values of 5 microM and 1 mM for the heterotrimer and the single alpha1' chain, respectively, which compare well with those reported for collagen type I (approximately 1 microM), gelatine (approximately 10 microM) and for octapeptides of the cleavage sequence (> or = 1 mM). The high affinity of the MMP-8 for the triple-helical heterotrimer and the fast digestion of this collagenous peptide confirm the decisive role of the hemopexin domain in recognition and possibly, partial unfolding of collagen.

Amino Acid Sequence↗
PubMed1996

X-ray structure of active site-inhibited clotting factor Xa. Implications for drug design and substrate recognition.

The 3.0-A resolution x-ray structure of human des-Gla-coagulation factor Xa (fXa) has been determined in complex with the synthetic inhibitor DX-9065a. The binding geometry is characterized primarily by two interaction sites: the naphthamidine group is fixed in the S1 pocket by a typical salt bridge to Asp-189, while the pyrrolidine ring binds in the unique aryl-binding site (S4) of fXa. Unlike the large majority of inhibitor complexes with serine proteinases, Gly-216 (S3) does not contribute to hydrogen bond formation. In contrast to typical thrombin binding modes, the S2 site of fXa cannot be used by DX-9065a since it is blocked by Tyr-99, and the aryl-binding site (S4) of fXa is lined by carbonyl oxygen atoms that can accommodate positive charges. This has implications for natural substrate recognition as well as for drug design.

Anticoagulants↗
PubMed1996

Enzyme flexibility, solvent and 'weak' interactions characterize thrombin-ligand interactions: implications for drug design.

BACKGROUND: The explosive growth in the rate of X-ray determination of protein structures is fuelled largely by the expectation that structural information will be useful for pharmacological and biotechnological applications. For example, there have been intensive efforts to develop orally administrable antithrombotic drugs using information about the crystal structures of blood coagulation factors, including thrombin. Most of the low molecular weight thrombin inhibitors studied so far are based on arginine and benzamidine. We sought to expand the database of information on thrombin-inhibitor binding by studying new classes of inhibitors. RESULTS: We report the structures of three new inhibitors complexed with thrombin, two based on 4-aminopyridine and one based on naphthamidine. We observe several geometry changes in the protein main chain and side chains which accompany inhibitor binding. The two inhibitors based on 4-aminopyridine bind in notably different ways: one forms a water-mediated hydrogen bond to the active site Ser195, the other induces a rotation of the Ser214-Trp215 peptide plane that is unprecedented in thrombin structures. These binding modes also differ in their 'weak' interactions, including CH-O hydrogen bonds and interactions between water molecules and aromatic pi-clouds. Induced-fit structural changes were also seen in the structure of the naphthamidine inhibitor complex. CONCLUSIONS: Protein flexibility and variable water structures are essential elements in protein-ligand interactions. Ligand design strategies that fail to take this into account may overlook or underestimate the potential of lead structures. Further, the significance of 'weak' interactions must be considered both in crystallographic refinement and in analysis of binding mechanisms.

4-Aminopyridine↗
PubMed1996

The ornithodorin-thrombin crystal structure, a key to the TAP enigma?

Ornithodorin, isolated from the blood sucking soft tick Ornithodoros moubata, is a potent (Ki = 10(-12) M) and highly selective thrombin inhibitor. Internal sequence homology indicates a two domain protein. Each domain resembles the Kunitz inhibitor basic pancreatic trypsin inhibitor (BPTI) and also the tick anticoagulant peptide (TAP) isolated from the same organism. The 3.1 A crystal structure of the ornithodorin-thrombin complex confirms that both domains of ornithodorin exhibit a distorted BPTI-like fold. The N-terminal portion and the C-terminal helix of each domain are structurally very similar to BPTI, whereas the regions corresponding to the binding loop of BPTI adopt different conformations. Neither of the two 'reactive site loops' of ornithodorin contacts the protease in the ornithodorin-thrombin complex. Instead, the N-terminal residues of ornithodorin bind to the active site of thrombin, reminiscent of the thrombin-hirudin interaction. The C-terminal domain binds at the fibrinogen recognition exosite. Molecular recognition of its target protease by this double-headed Kunitz-type inhibitor diverges considerably from other members of this intensely studied superfamily. The complex structure provides a model to explain the perplexing results of mutagenesis studies on the TAP-factor Xa interaction.

Amino Acid Sequence↗
PubMed1996

The 1.8 A crystal structure of human cathepsin G in complex with Suc-Val-Pro-PheP-(OPh)2: a Janus-faced proteinase with two opposite specificities.

The crystal structure of human neutrophil cathepsin G, complexed with the peptidyl phosphonate inhibitor Suc-Val-Pro-PheP-(OPh)2, has been determined to a resolution of 1.8 A using Patterson search techniques. The cathepsin G structure shows the polypeptide fold characteristic of trypsin-like serine proteinases and is especially similar to rat mast cell proteinase II. Unique to cathepsin G, however, is the presence of Glu226 (chymotrypsinogen numbering), which is situated at the bottom of the S1 specificity pocket, dividing it into two compartments. For this reason, the benzyl side chain of the inhibitor PheP residue does not fully occupy the pocket but is, instead, located at its entrance. Its positively charged equatorial edge is involved in a favourable electrostatic interaction with the negatively charged carboxylate group of Glu226. Arrangement of this Glu226 carboxylate would also allow accommodation of a Lys side chain in this S1 pocket, in agreement with the recently observed cathepsin G preference for Lys and Phe at P1. The cathepsin G complex with the covalently bound phosphonate inhibitor mimics a tetrahedral substrate intermediate. A comparison of the Arg surface distributions of cathepsin G, leukocyte elastase and rat mast cell protease II shows no simple common recognition pattern for a mannose-6-phosphate receptor-independent targeting mechanism for sorting of these granular proteinases.

Amino Acid Sequence↗
PubMed1996

The 2.3 A crystal structure of the catalytic domain of recombinant two-chain human tissue-type plasminogen activator.

Tissue-type plasminogen activator (t-PA), a multidomainal serine proteinase of the trypsin-family, catalyses the rate-limiting step in fibrinolysis, the activation of plasminogen to the fibrin-degrading proteinase plasmin. Trigonal crystals have been obtained of the recombinant catalytic domain of human-two-chain t-PA, consisting of a 17 residue A chain and the 252 residue B chain. Its X-ray crystal structure has been solved applying Patterson and isomorphous replacement methods, and has been crystallographically refined to an R-value of 0.184 at 2.3 A resolution. The chain fold, active-site geometry and Ile276-Asp477 salt bridge are similar to that observed for trypsin. A few surface-located insertion loops differ significantly, however. The disulfide bridge Cys315-Cys384, practically unique to the plasminogen activators, is incorporated without drastic conformational changes as the insertion loop preceding Cys384 makes a bulge on the molecular surface. The unique basic insertion loop Lys296-Arg304 flanking the primed subsites, which has been shown to be of importance for PAI-1 binding and for fibrin specificity, is partially disordered; it can therefore freely adapt to proteins docking to the active site. The S1 pocket of t-PA is almost identical to that of trypsin, whereas the S2 site is considerably reduced in size by the imposing Tyr368 side-chain, in agreement with the measured preference for P1 Arg and P2 Gly residues. The neighbouring S3-S4 hydrophobic groove is mainly hydrophobic in nature. The structure of the proteinase domain of two-chain t-PA suggests that the formation of a salt bridge between Lys429 and Asp477 may contribute to the unusually high catalytic activity of single-chain t-PA, thus stabilizing the catalytically active conformation without unmasking the Ile276 amino terminus. Modeling studies show that the covalently bound kringle 2 domain in full-length t-PA could interact with an extended hydrophobic groove in the catalytic domain; in such a docking geometry its "lysine binding site" and the "fibrin binding patch" of the catalytic domain are in close proximity.

Amino Acid Sequence↗