A stoichiometric complex formed between human alpha‐thrombin and D‐Phe‐Pro‐Arg chloromethylketone was crystallized in an orthorhombic crystal form. Orientation and position of a starting model derived from homologous modelling were determined by Patterson search methods. The thrombin model was completed in a cyclic modelling‐crystallographic refinement procedure to a final R‐value of 0.171 for X‐ray data to 1.92 A. The structure is in full agreement with published cDNA sequence data. The A‐chain, ordered only in its central part, is positioned along the molecular surface opposite to the active site. The B‐chain exhibits the characteristic polypeptide fold of trypsin‐like proteinases. Several extended insertions form, however, large protuberances; most important for interaction with macromolecular substrates is the characteristic thrombin loop around Tyr60A‐Pro60B‐Pro60C‐Trp60D (chymotrypsinogen numbering) and the enlarged loop around the unique Trp148. The former considerably restricts the active site cleft and seems likely to be responsible for poor binding of most natural proteinase inhibitors to thrombin. The exceptional specificity of D‐Phe‐Pro‐Arg chloromethylketone can be explained by a hydrophobic cage formed by Ile174, Trp215, Leu99, His57, Tyr60A and Trp60D. The narrow active site cleft, with a more polar base and hydrophobic rims, extends towards the arginine‐rich surface of loop Lys70‐Glu80 that probably represents part of the anionic binding region for hirudin and fibrinogen.

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 crystallographic structure of a recombinant hirudin-thrombin complex has been solved at 2.3 angstrom (A) resolution. Hirudin consists of an NH2-terminal globular domain and a long (39 A) COOH-terminal extended domain. Residues Ile1 to Tyr3 of hirudin form a parallel beta-strand with Ser214 to Glu217 of thrombin with the nitrogen atom of Ile1 making a hydrogen bond with Ser195 O gamma atom of the catalytic site, but the specificity pocket of thrombin is not involved in the interaction. The COOH-terminal segment makes numerous electrostatic interactions with an anion-binding exosite of thrombin, whereas the last five residues are in a helical loop that forms many hydrophobic contacts. In all, 27 of the 65 residues of hirudin have contacts less than 4.0 A with thrombin (10 ion pairs and 23 hydrogen bonds). Such abundant interactions may account for the high affinity and specificity of hirudin.

The substrate-like 'canonical' inhibition by the 'small' serine proteinase inhibitors and the productlike inhibition by the carboxypeptidase inhibitor have provided the only atomic models of protein inhibitor -proteinase interactions for about 15 years. The recently published structures of cystatin/ stefin -papain complexes and of hirudin -thrombin complexes reveal novel non-substrate-like interactions, In addition, the structure of pro-carboxypeptidase showes a mode of inactivation which bears resemblance to proteinase/protein inhibitor systems. Considerable progress in understanding the transition between native and cleaved states of the serpins has also been made by several recent structural studies.Proteinase inhibitors are important tools of nature for regulating the proteolytic activity of their target proteinases, for blocking these in emergency cases, or for signaling receptor interactions or clearance. Endogenous inhibitors appear to be always proteins; small non-proteinaceous inhibitors which impair the proteolytic activity of host proteinases are produced in microorganisms.The number of proteinaceous proteinase inhibitors isolated and identified so far is extremely large. In a now classical review paper, Laskowski and Kato [2] introduced for the first time a rational nomenclature by grouping these diverse inhibitors into distinct protein families. In the meantime, this list of families has considerably expanded with the advent of many new inhibitor species, and is still growing.The majority of protein inhibitors known and characterized so far are directed towards serine proteinases. Within the last few years, a large number of protein inhibitors of cysteine proteinases have also been discovered and characterized [3,4]. In contrast, only a few protein inhibitors directed towards metallo-proteinases (TIMP and PCI, see [5,61) or aspartyl proteinases (see [7][8][9]) are known to date. The azmacroglobulin family presents an exception, as these proteins can inhibit all of these proteinases according to a 'molecular trap' mechanism by virtue of a promiscuous 'bait region' (see [IOI).Until recently, X-ray crystal structures of only a few serine proteinase inhibitors, one carboxypeptidase inhibitor, and some of their complexes with cognate proteinases were available. The X-ray crystal structures of protein inhibitors published up to 1985 have been reviewed by Read and James A new aspect is provided by inhibitor structures elucidated by two-dimensional NMR methods (see [22, 231 for reviews). These data are often complementary to X-ray data, but are restricted to isolated inhibitors of relatively small molecular mass; until now, no NMR structure of a protein inhibitor has been reported for whch there is no X-ray structure available.In this review, we shall attempt to illuminate the characteristic structural properties conferring inhibitory activity to proteins. Nature has used diverse approaches to achieve proteinase inhibition. This is particularly well illustrated by some more recently published structures. In...

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.