Thrombin is a multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X-ray crystal structure of human alpha-thrombin was determined in its complex with the specific thrombin inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467-3475). The crystallographic refinement of the PPACK-thrombin model has now been completed at an R value of 0.156 (8 to 1.92 A); in particular, the amino- and the carboxy-termini of the thrombin A-chain are now defined and all side-chain atoms localized; only proline 37 was found to be in a cis-peptidyl conformation. The thrombin B-chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymotrypsin with a root-mean-square (r.m.s.) deviation of 0.8 A for their alpha-carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A-chain is arranged in a boomeranglike shape against the B-chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding. Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the carboxy-terminal B-chain helix (forming the presumed heparin-binding site) and on the surface of loop segment 70-80 (the fibrin[ogen] secondary binding exosite), respectively; the negatively charged residues are more clustered in the ringlike region between both poles, particularly around the active site. Several of the charged residues are involved in salt bridges; most are on the surface, but 10 charged protein groups form completely buried salt bridges and clusters. These electrostatic interactions play a particularly important role in the intrachain stabilization of the A-chain, in the coherence between the A- and the B-chain, and in the surface structure of the fibrin(ogen) secondary binding exosite (loop segment 67-80).(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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The role of electrostatic interactions in stabilization of the thrombin-hirudin complex has been investigated by means of two macroscopic approaches: the modified Tanford-Kirkwood model and the finite-difference method for numerical solution of the Poisson-Boltzmann equations. The electrostatic potentials around the thrombin and hirudin molecules were asymmetric and complementary, and it is suggested that these fields influence the initial orientation in the process of the complex formation. The change of the electrostatic binding energy due to mutation of acidic residues in hirudin has been calculated and compared with experimentally determined changes in binding energy. In general, the change in electrostatic binding energy for a particular mutation calculated by the modified Tanford-Kirkwood approach agreed well with the experimentally observed change. The finitedifference approach tended to overestimate changes in binding energy when the mutated residues were involved in short-range electrostatic interactions. Decreases in binding energy caused by mutations of amino acids that do not make any direct ionic interactions (e.g., Glu 61 and Glu 62 of hirudin) can be explained in terms of the interaction of these charges with the positive electrostatic potential of thrombin. Differences between the calculated and observed changes in binding energy are discussed in terms of the crystal structure of the thrombin-hirudin complex.Keywords: electrostatic interactions; hirudin; protein-protein interactions; thrombin Thrombin is a serine protease that plays a central role in blood coagulation. It cleaves fibrinogen to yield fibrin monomers that form the basis of the blood clot. In addition, thrombin activates a number of other proteins involved in coagulation. Thrombin can be distinguished from other serine proteases, such as trypsin, in that it achieves its specificity by using binding sites, called exosites, that are distant from the catalytic center (Fenton, 1988). The crystal structures of thrombin (Bode et al., 1989(Bode et al., , 1992 and its complexes with the polypeptide inhibitor hirudin (Kinemage 1; Griitter et al., 1990;Rydel et al., 1990Rydel et al., , 1991 cleft of thrombin, the C-terminal region binds to a positively charged surface groove called the fibrinogen-recognition exosite. The C-terminal region of recombinant hirudin (rhir) between residues 55 and 65 contains five negatively charged residues, and results from studies using site-directed mutagenesis indicate that each of these negatively charged residues (Asp 55: Glu 57: Glu 58: Glu 61', and Glu 62') contributes to the stabilization of the complex (Braun et al., 1988a;Betz et al., 1991). In addition, the effect of ionic strength on the interaction between thrombin and hirudin suggests that electrostatic interactions with the C-terminal region of hirudin are important for the rate of complex formation and the stability of the complex (Stone et al., 1989). However, although protein engineering studies indicate that each of the negatively char...

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