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.
From the lysosomal cysteine proteinase cathepsin B, isolated from human liver in its two‐chain form, monoclinic crystals were obtained which contain two molecules per asymmetric unit. The molecular structure was solved by a combination of Patterson search and heavy atom replacement methods (simultaneously with rat cathepsin B) and refined to a crystallographic R value of 0.164 using X‐ray data to 2.15 A resolution. The overall folding pattern of cathepsin B and the arrangement of the active site residues are similar to the related cysteine proteinases papain, actinidin and calotropin DI. 166 alpha‐carbon atoms out of 248 defined cathepsin B residues are topologically equivalent (with an r.m.s. deviation of 1.04 A) with alpha‐carbon atoms of papain. However, several large insertion loops are accommodated on the molecular surface and modify its properties. The disulphide connectivities recently determined for bovine cathepsin B by chemical means were shown to be correct. Some of the primed subsites are occluded by a novel insertion loop, which seems to favour binding of peptide substrates with two residues carboxy‐terminal to the scissile peptide bond; two histidine residues (His110 and His111) in this “occluding loop' provide positively charged anchors for the C‐terminal carboxylate group of such polypeptide substrates. These structural features explain the well‐known dipeptidyl carboxypeptidase activity of cathepsin B. The other subsites adjacent to the reactive site Cys29 are relatively similar to papain; Glu245 in the S2 subsite favours basic P2‐side chains. The above mentioned histidine residues, but also the buried Glu171 might represent the group with a pKa of approximately 5.5 near the active site, which governs endo‐ and exopeptidase activity. The “occluding loop' does not allow cystatin‐like protein inhibitors to bind to cathepsin B as they do to papain, consistent with the reduced affinity of these protein inhibitors for cathepsin B compared with the related plant enzymes.
The structure of the ternary complex of human a-thrombin with a covalently bound analogue of fibrinopeptide A and a C-terminal hirudin peptide has been determined by X-ray diffraction methods at 0.25 nm resolution. Fibrinopeptide A folds in a compact manner, bringing together hydrophobic residues that slot into the apolar binding site of human a-thrombin. Fibrinogen residue Phe8 occupies the aryl-binding site of thrombin, adjacent to fibrinogen residues Leu9 and Val15 in the S2 subsite. The species diversity of fibrinopeptide A is analysed with respect to its conformation and its interaction with thrombin. The non-covalently attached peptide fragment hirudin(54 -65) exhibits an identical conformation to that observed in the hirudin-thrombin complex. The occupancy of the secondary fibrinogen-recognition exosite by this peptide imposes restrictions on the manner of fibrinogen binding. The surface topology of the thrombin molecule indicates positions PI' -P3', differ from those of the canonical serine-proteinase inhibitors, suggesting a mechanical model for the switching of thrombin activity from fibrinogen cleavage to protein-C activation on thrombomodulin complex formation. The multiple interactions between thrombin and fibrinogen provide an explanation for the narrow specificity of thrombin. Structural grounds can be put forward for certain congenital clotting disorders.The specific cleavage of fibrinogen by the serine proteinase thrombin initiates the polymerisation of fibrin monomers, a primary event in blood clot formation [4]. Fibrinogen (340 kDa) is a covalently linked dimer of three peptide chains, with stoichiometry (Aa, BP,y), [5]. The cleavage releases two peptides, fibrinopeptides A and B, from the N-termini of chains Aa and Bfl respectively, thereby revealing recognition sites for aggregation with the y chain.Thrombin exhibits primarily a trypsin-like specificity, i.e. a preference for P1 arginine residues [6]. The cleavage of fibrinogen by thrombin represents a very specific reaction however; of the 376 Arg/Lys-Xaa bonds in the fibrinogen molecule, thrombin cleaves only four, releasing the fibrinopeptides [6]. Residues of fibrinogen contributing to this exceptional specificity have been localised to the first 51 amino acids of the Acc chain [7, 81. This region has been further dissected to explore subsites, assigning roles (in order of im-Enzyrnr. Thrombin (EC 3.4 .21 .S).Nomencluture. The peptide and subsite nomenclature is that suggested by Schechter and Berger 111: amino acid residues of substrates are numbered P1, P2, P3 etc. towards the amino terminus, and P1 ', P2', P3' etc. towards the carboxy terminus from the reactive-sitc bond; the complementary subsites of the enzyme are numbered S1, S2, S3 etc. and SI', S2', S3' etc., respectively. Thrombin residues are numbered according to the chymotrypsinogen system [2], with inserted residues marked by a lower-case suffix [ 3 ] . Fibrinogen residues are prefixed by the letter F, hirudin residues by the letter H and uPhe-Pro-Arg-MeC1 residues by the le...
Crystal structure analysis and refinement at 2.0 A resolution of a rhombohedral crystal form of human annexin V at high calcium concentration revealed a domain motion compared to the previously analysed hexagonal crystal form. Five calcium ions were located on the convex face of the molecule. Three strongly bound calciums are liganded at protruding interhelical loops and Asp or Glu residues in homologous positions in repeats I, II and IV. Five proteinaceous oxygens and one solvent molecule form the coordination polyhedron in each case. The unoccupied seventh site is suggested as the phospholipid headgroup binding site. Two more weakly bound sites were identified by lanthanum labelling. The structural features suggest that annexin V attaches with its convex face to membranes by specified calcium mediated interactions with at least three phospholipids. The adjacent membrane bilayer may thus become locally disordered and permeable to allow calcium inflow through the central polar channel of the molecule.
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