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 2.0 A X-ray crystal structure of the snake venom zinc-endopeptidase adamalysin II. Primary and tertiary structure determination, refinement, molecular structure and comparison with astacin, collagenase and thermolysin.

Adamalysin II, alias proteinase II, a 24 kDa zinc-endopeptidase isolated from the snake venom of the Eastern diamondback rattlesnake Crotalus adamanteus, is a prototype of the proteolytic domain of snake venom metalloproteinases and of domains found in mammalian reproductive tract proteins. Its 2.0 A crystal and molecular structure was solved by multiple isomorphous replacement using six heavy-atom derivatives, and was refined to a crystallographic R-value of 0.172. 201 of the 203 amino acid residues of adamalysin II are defined by electron density; only the first two residues are disordered and crystallographically undefined in the crystal structure. Three-quarters of these crystallographic amino acid residue assignments were confirmed by chemical sequencing. In addition, the active-site zinc-ion, a hepta-coordinated calcium ion, a fixed sulphate anion and 173 solvent molecules were localized in the structure. Adamalysin II is an ellipsoidal molecule with a relatively flat active-site cleft separating the "upper" main body from a small "lower" subdomain. The regularly folded N-terminal upper domain consists essentially of a central, highly twisted five-stranded beta-pleated sheet flanked by a long and a short surface located helix on its convex side, and by two long helices, one of which represents the central "active site helix", on its concave side. The lower subdomain, comprising the last 50 residues, is organized in multiple turns, with the chain ending in a long C-terminal helix and an extended segment clamped to the upper domain via a disulphide bridge. The catalytic zinc-ion, located at the bottom of the active-site cleft, is almost tetrahedrally co-ordinated by His142, His146 and His152, and a water molecule anchored to an intermediate glutamic acid residue (Glu143), with the three imidazole N epsilon 2 nitrogen atoms 2.1 A and the solvent oxygen atom 2.4 A away from the zinc ion. His142, Glu143 and His146 are part of the long active-site helix, which extends up to Gly149, where it turns sharply away towards His152. The importance of these residues for structure and activity of adamalysin II explains their occurrence in the HEXXHXXGXXH consensus sequence. Asp153, which is strictly conserved in these snake venom and reproductive tract metalloproteinases, is buried in the subdomain and seems to stabilize the hydrophobic active-site basement. Some residues behind, the adamalysin peptide chain folds into a characteristic 1,4-turn (the "Met-turn") containing the conserved Met166, which forms a hydrophobic basement for the three zinc-binding imidazoles.(ABSTRACT TRUNCATED AT 400 WORDS)

The interaction of thrombin with fibrinogen. A structural basis for its specificity.

The structure of the ternary complex of human alpha-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 alpha-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 P1'-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 refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity.

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 calcium binding sites in human annexin V by crystal structure analysis at 2.0 A resolution. Implications for membrane binding and calcium channel activity.

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 specific 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.

Crystals of the NC1 domain of human type IV collagen.

Crystals of the non-collagenous C-terminal region (NC1) of type IV collagen have been obtained from human placenta. These crystals diffract to 2.0 A, and belong to space group P22(1)2(1), with cell dimensions a = 81 A, b = 158 A, c = 138 A, alpha = beta = gamma = 90 degrees. The crystals contain one hexamer in the asymmetric unit; they are very stable with respect to X-rays.

The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment.

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.

The refined 2.3 A crystal structure of human leukocyte elastase in a complex with a valine chloromethyl ketone inhibitor.

The stoichiometric complex formed between human leukocyte elastase and a synthetic MeO-Suc-Ala-Ala-Pro-Val chloromethyl ketone inhibitor was co-crystallized and its X-ray structure determined, using Patterson search methods. Its structure has been crystallographically refined to a final R value of 0.145 (8.0 and 2.3 A). The enzyme structure is very similar to that recently observed in a complex formed with the ovomucoid third domain from turkey [(1986) EMBO J. 5,2453-2458]. The rms deviation of all alpha-carbon atoms is 0.32 A. The peptidic inhibitor is bound in a similar overall conformation as the ovomucoid binding segment. Covalent bonds are formed between Val-P1 of the inhibitor and His-57 NE2 and Ser-195 OG of the enzyme. The carbonyl carbon is tetrahedrally deformed to a hemiketal. The valine side chain is arranged in the S1 pocket in the g-conformation.

Molecular structure of the bilin binding protein (BBP) from Pieris brassicae after refinement at 2.0 A resolution.

The bilin binding protein (BBP) from the insect Pieris brassicae has been analysed for amino acid sequence, spectral properties and three-dimensional structure. The crystal structure that had been determined by isomorphous replacement has been refined at 2.0 A (1 A = 0.1 nm) resolution to an R-value of 0.20. The asymmetric unit contains four independent subunits of BBP. The co-ordinate differences are 0.25 A, in accord with the estimated error in co-ordinates. The polypeptide chain fold is characterized by an eight-stranded barrel. The connecting loops splay out at the upper end of the barrel and open it, whilst the lower end is closed. The overall shape resembles a calyx. The biliverdin IX gamma chromophore is located in a central cleft at the upper end of the barrel. The bilatriene moiety is in cyclic helical geometry with configuration Z,Z,Z and conformation syn,syn,syn. The geometry is in accord with the spectral properties and permits a correlation between sign of the circular dichroism bands and sense of the bilatriene helices. The fold of BBP is related to retinol binding protein (RBP), as had been recognized in the preliminary analysis, although the amino acid sequences of RBP and BBP show only 10% homology. There are large differences in the loops at the upper end of the barrel, whilst the segments of the centre and the lower end of the barrel superimpose closely. The ligands of BBP and RBP, biliverdin and retinol, respectively, are also similarly located.

Crystallization, crystal structure analysis and preliminary molecular model of the bilin binding protein from the insect Pieris brassicae.

The bilin binding protein of the butterfly Pieris brassicae has been prepared, crystallized and its crystal structure determined at high resolution using film and FAST area detector intensity data. The crystallographic asymmetric unit contains a tetramer of identical subunits with a molecular weight of about 90,000. The crystal structure was determined by isomorphous replacement. Use was made of the molecular symmetry to improve phases. A molecular interpretation of the electron density distribution and partial tracing of the polypeptide chain was possible without amino acid sequence information, as the fold is very similar to retinol binding protein. It is characterized by a beta-barrel formed by two orthogonal beta-sheets and an alpha-helix. The bilin pigment seems to be bound within the beta-barrel analogously to retinol in retinol binding protein. The tetramer in the crystal has C2 symmetry and is a dimer of dimers of quasi-equivalent subunits.

Epithelial blood group antigens in colon polyps. I. Morphologic distribution and relationship to differentiation.

The distribution patterns of epithelial blood group AB antigens (BG) in colon polyps of varying degrees of differentiation were studied by the mixed cell agglutination reaction. BG appeared in colon polyps if a certain degree of dedifferentiation was present. Two different distribution patterns were recognized: 1) association of BG with the secretory part of goblet cells with slight-to-moderate atypia and 2) association of BG with the whole cells in cases of pronounced dedifferentiation and loss of secretory activity. The second type resembled the distribution pattern of BG found previously in colon carcinomas. With respect to BG, the mucosa of colon polyps behaved similarly to embryonal colon mucosa. The detection of BG represents a useful method to assess objectively the degree of dedifferentiation in most colon polyps.