Wolfram Bode

Mechanism of inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1.

Matrix metalloproteinases (MMPs) are zinc endopeptidases that are required for the degradation of extracellular matrix components during normal embryo development, morphogenesis and tissue remodelling. Their proteolytic activities are precisely regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in diseases such as arthritis, atherosclerosis, tumour growth and metastasis. Here we report the crystal structure of an MMP-TIMP complex formed between the catalytic domain of human stromelysin-1 (MMP-3) and human TIMP-1. TIMP-1, a 184-residue protein, has the shape of an elongated, contiguous wedge. With its long edge, consisting of five different chain regions, it occupies the entire length of the active-site cleft of MMP-3. The central disulphide-linked segments Cys 1-Thr 2-Cys 3-Val 4 and Ser 68-Val 69 bind to either side of the catalytic zinc. Cys 1 bidentally coordinates this zinc, and the Thr-2 side chain extends into the large specificity pocket of MMP-3. This unusual architecture of the interface between MMP-3 and TIMP-1 suggests new possibilities for designing TIMP variants and synthetic MMP inhibitors with potential therapeutic applications.

The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases.

The crystal structure of chicken egg white cystatin has been solved by X‐ray diffraction methods using the multiple isomorphous replacement technique. Its structure has been refined to a crystallographic R value of 0.19 using X‐ray data between 6 and 2.0A. The molecule consists mainly of a straight five‐turn alpha‐helix, a five‐stranded antiparallel beta‐pleated sheet which is twisted and wrapped around the alpha‐helix and an appending segment of partially alpha‐helical geometry. The ‘highly conserved’ region from Gln53I to Gly57I implicated with binding to cysteine proteinases folds into a tight beta‐hairpin loop which on opposite sides is flanked by the amino‐terminal segment and by a second hairpin loop made up of the similarly conserved segment Pro103I ‐ Trp104I. These loops and the amino‐terminal Gly9I ‐ Ala10I form a wedge‐shaped ‘edge’ which is quite complementary to the ‘active site cleft’ of papain. Docking experiments suggest a unique model for the interaction of cystatin and papain: according to it both hairpin loops of cystatin make major binding interactions with the highly conserved residues Gly23, Gln19, Trp177 and Ala136 of papain in the neighbourhood of the reactive site Cys25; the amino‐terminal segment Gly9I ‐ Ala10I of bound cystatin is directed towards the substrate subsite S2, but in an inappropriate conformation and too far away to be attacked by the reactive site Cys25. As a consequence, the mechanism of the interaction between cysteine proteinases and their cystatin‐like inhibitors seems to be fundamentally different from the ‘standard mechanism’ defined for serine proteinases and most of their protein inhibitors.

The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction.

A stoichiometric complex of human stefin B and carboxymethylated papain has been crystallized in a trigonal crystal form. Data to 2.37 A resolution were collected using the area detector diffractometer FAST. The crystal structure of the complex has been solved by Patterson search techniques using papain as search model. Starting from the structure of chicken cystatin, the stefin structure was elucidated through cycles of model building and crystallographic refinement. The current crystallographic R factor is 0.19. Like cystatin, the stefin molecule consists of a five stranded beta‐sheet wrapped around a five turn alpha‐helix, but with an additional carboxy terminal strand running along the convex side of the sheet. Topological equivalence of stefin and cystatin reveal the previous sequence alignment to be incorrect in part, through deletion of the intermediate helix. The conserved residues form a tripartite wedge, which slots into the papain active site as proposed through consideration of the tertiary structures of the individual components (Bode et al., 1988). The main interactions are provided by the amino terminal ‘trunk’ (occupying the ‘unprimed’ subsites of the enzyme), and by the first hairpin loop, containing the highly conserved QVVAG sequence, with minor contributions from the second hairpin loop. The carboxyl terminus of stefin provides an additional interaction region with respect to cystatin. The interaction is dominated by hydrophobic contacts. Inhibition by the cysteine proteinase inhibitors is fundamentally different to that observed for the serine proteinase inhibitors.

The X-ray crystal structure of the catalytic domain of human neutrophil collagenase inhibited by a substrate analogue reveals the essentials for catalysis and specificity.

Matrix metalloproteinases are a family of zinc endopeptidases involved in tissue remodelling. They have been implicated in various disease processes including tumour invasion and joint destruction. These enzymes consist of several domains, which are responsible for latency, catalysis and substrate recognition. Human neutrophil collagenase (PMNL‐CL, MMP‐8) represents one of the two ‘interstitial’ collagenases that cleave triple helical collagens types I, II and III. Its 163 residue catalytic domain (Met80 to Gly242) has been expressed in Escherichia coli and crystallized as a non‐covalent complex with the inhibitor Pro‐Leu‐Gly‐hydroxylamine. The 2.0 A crystal structure reveals a spherical molecule with a shallow active‐site cleft separating a smaller C‐terminal subdomain from a bigger N‐terminal domain, composed of a five‐stranded beta‐sheet, two alpha‐helices, and bridging loops. The inhibitor mimics the unprimed (P1‐P3) residues of a substrate; primed (P1′‐P3′) peptide substrate residues should bind in an extended conformation, with the bulky P1′ side‐chain fitting into the deep hydrophobic S1′ subsite. Modelling experiments with collagen show that the scissile strand of triple‐helical collagen must be freed to fit the subsites. The catalytic zinc ion is situated at the bottom of the active‐site cleft and is penta‐coordinated by three histidines and by both hydroxamic acid oxygens of the inhibitor. In addition to the catalytic zinc, the catalytic domain harbours a second, non‐exchangeable zinc ion and two calcium ions, which are packed against the top of the beta‐sheet and presumably function to stabilize the catalytic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor rhodniin in complex with thrombin.

Rhodniin is a highly specific inhibitor of thrombin isolated from the assassin bug Rhodnius prolixus. The 2.6 Angstrum crystal structure of the non‐covalent complex between recombinant rhodniin and bovine alpha‐thrombin reveals that the two Kazal‐type domains of rhodniin bind to different sites of thrombin. The amino‐terminal domain binds in a substrate‐like manner to the narrow active‐site cleft of thrombin; the imidazole group of the P1 His residue extends into the S1 pocket to form favourable hydrogen/ionic bonds with Asp189 at its bottom, and additionally with Glu192 at its entrance. The carboxy‐terminal domain, whose distorted reactive‐site loop cannot adopt the canonical conformation, docks to the fibrinogen recognition exosite via extensive electrostatic interactions. The rather acidic polypeptide linking the two domains is displaced from the thrombin surface, with none of its residues involved in direct salt bridges with thrombin. The tight (Ki = 2 × 10(‐13) M) binding of rhodniin to thrombin is the result of the sum of steric and charge complementarity of the amino‐terminal domain towards the active‐site cleft, and of the electrostatic interactions between the carboxy‐terminal domain and the exosite.

Mechanism of inhibition of papain by chicken egg white cystatin. Inhibition constants of N-terminally truncated forms and cyanogen bromide fragments of the inhibitor.

N‐terminally truncated forms of chicken egg white cystatin and its cyanogen bromide fragments were isolated and assayed for inhibition of papain. Truncated forms beginning with Gly‐9 and Ala‐10 had a 5000‐fold lower affinity for papain than the two isoelectric forms (pI=6.5 and 5.6) of the full‐length inhibitor (K i=6 pM and 7 pM) or a truncated form beginning with Leu‐7 (K i=6 pM), indicating the outstanding importance of one or two residues preceding conserved Gly‐9 for binding. A weak inhibition of papain (K i=900 nM) was exhibited by the intermediate cyanogen bromide fragment (residues 30–89) containing the chicken cystatin QLVSG variation of the QVVAG segment which is conserved in almost all members of the cystatin superfamily. The obtained affiffity data provide independent evidence for the validity of the proposed docking model of a chicken cystatin‐papain complex [(1988) EMBO J. 7, 2593–2599].

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.

Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin.

The crystal structure of the complex formed between eglin c, an elastase inhibitor from the medical leech, and subtilisin Carlsberg has been determined at 1.2 A resolution by a combination of Patterson search methods and isomorphous replacement techniques. The structure has been refined to a crystallographic R‐value of 0.18 (8‐1.2 A). Eglin consists of a four‐stranded beta‐sheet with an alpha‐helical segment and the protease‐binding loop fixed on opposite sides. This loop, which contains the reactive site Leu45I‐‐Asp46I, is mainly held in its conformation by unique electrostatic/hydrogen bond interactions of Thr44I and Asp46I with the side chains of Arg53I and Arg51I which protrude from the hydrophobic core of the molecule. The conformation around the reactive site is similar to that found in other proteinase inhibitors. The nine residues of the binding loop Gly40I‐‐Arg48I are involved in direct contacts with subtilisin. In this interaction, eglin segment Pro42I‐‐Thr44I forms a three‐stranded anti‐parallel beta‐sheet with subtilisin segments Gly100‐‐Gly102 and Ser125‐‐Gly127. The reactive site peptide bond of eglin is intact, and Ser221 OG of the enzyme is 2.81 A apart from the carbonyl carbon.

Crystal structure of bovine trypsinogen at 1.8 Å resolution. I. Data collection, application of Patterson search techniques and preliminary structural interpretation

Abstract X-ray intensity data to 1.8 A resolution were collected from native trigonal crystals of bovine trypsinogen. The orientation and position of the trypsinogen molecules within their crystal cells were determined by Patterson search techniques using the refined model of bovine trypsin (Bode & Schwager, 1975), and by subsequent R factor refinement. The translation functions allowed discrimination between the enantiomorphic space groups P 3 2 21 and P 3 1 21. After one constrained crystallographic refinement cycle, which reduced the crystallographic reliability factor ( R ) from 35% to 31%, a preliminary difference Fourier map showed several interesting details. Several refinement cycles reduced the value of R to 23%. The overall chain folding is very similar to trypsin. The chain segments, including residues 184 to 193 † and 217 to 223, which form the specificity pocket in trypsin, are flexible in trypsinogen. The autolysis loop is partially mobile between residues 142 and 152. There is no continuing electron density for the N terminal residues preceding Tyr20. This indicates that the N terminus may be only weakly fixed to the rest of the molecule or may even float freely in solution.