Structure of tick anticoagulant peptide at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor.

The structure of tick anticoagulant peptide (TAP) has been determined by X-ray crystallography at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP-BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 A, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c-axis with a diameter of about 45 A. Individual TAP units interact in a head-to-tail fashion, the positive end of one molecule associating with the distal negative end of another, and vice versa. The BPTI molecules have a uniformly distributed positively charged surface that interacts extensively through 14 hydrogen bonds and two hydrogen bonded salt bridges with the helical groove around the helical TAP chains. Comparing the structure of TAP in TAP-BPTI with TAP bound to factor Xa(Xa) suggests a massive reorganization in the N-terminal tetrapeptide and the first disulfide loop of TAP (Cys5T-Cys15T) upon binding to Xa. The Tyr1(T)OH atom of TAP moves 14.2 A to interact with Asp189 of the S1 specificity site, Arg3(T)CZ moves 5.0 A with the guanidinium group forming a cation-pi-electron complex in the S4 subsite of Xa, while Lys7(T)NZ differs in position by 10.6 A in TAP-BPTI and TAP-Xa, all of which indicates a different pre-Xa-bound conformation for the N-terminal of TAP in its native state. In contrast to TAP, the BPTI structure of TAP-BPTI is practically the same as all those of previously determined structures of BPTI, only arginine and lysine side-chain conformations showing significant differences.

Bound structures of novel P3-P1' beta-strand mimetic inhibitors of thrombin.

The X-ray crystal structures of four beta-strand-templated active site inhibitors of thrombin containing P1' groups have been determined and refined at about 2.1-A resolution to crystallographic R-values between 0.148 and 0.164. Two of the inhibitors have an alpha-ketoamide functionality at the scissile bond; the other two have a nonhydrolyzable electrophilic group at the P1' position. The binding of lysine is compared with that of arginine at the S1 specificity site, while that of D,L-phenylalanine enantiomorphs is compared in the S3 region of thrombin. Four different P1' moieties bind at the S1' subsite in three different ways. The binding constants vary between 2.0 microM and 70 pM. The bound structures are used to intercorrelate the various binding constants and also lead to insightful inferences concerning binding at the S1' site of thrombin.

Structure of extracellular tissue factor complexed with factor VIIa inhibited with a BPTI mutant.

The event that initiates the extrinsic pathway of blood coagulation is the association of coagulation factor VIIa (VIIa) with its cell-bound receptor, tissue factor (TF), exposed to blood circulation following tissue injury and/or vascular damage. The natural inhibitor of the TF.VIIa complex is the first Kunitz domain of tissue factor pathway inhibitor (TFPI-K1). The structure of TF. VIIa reversibly inhibited with a potent (Ki=0.4 nM) bovine pancreatic trypsin inhibitor (BPTI) mutant (5L15), a homolog of TFPI-K1, has been determined at 2.1 A resolution. When bound to TF, the four domain VIIa molecule assumes an extended conformation with its light chain wrapping around the framework of the two domain TF cofactor. The 5L15 inhibitor associates with the active site of VIIa similar to trypsin-bound BPTI, but makes several unique interactions near the perimeter of the site that are not observed in the latter. Most of the interactions are polar and involve mutated positions of 5L15. Of the eight rationally engineered mutations distinguishing 5L15 from BPTI, seven are involved in productive interactions stabilizing the enzyme-inhibitor association with four contributing contacts unique to the VIIa.5L15 complex. Two additional unique interactions are due to distinguishing residues in the VIIa sequence: a salt bridge between Arg20 of 5L15 and Asp60 of an insertion loop of VIIa, and a hydrogen bond between Tyr34O of the inhibitor and Lys192NZ of the enzyme. These interactions were used further to model binding of TFPI-K1 to VIIa and TFPI-K2 to factor Xa, the principal activation product of TF.VIIa. The structure of the ternary protein complex identifies the determinants important for binding within and near the active site of VIIa, and provides cogent information for addressing the manner in which substrates of VIIa are bound and hydrolyzed in blood coagulation. It should also provide guidance in structure-aided drug design for the discovery of potent and selective small molecule VIIa inhibitors.

Molecular model of human beta-cell glucokinase built by analogy to the crystal structure of yeast hexokinase B.

Recent studies have shown that mutations in human beta-cell glucokinase that impair the activity of this key regulatory enzyme of glycolysis can cause early-onset non-insulin-dependent diabetes mellitus (NIDDM). The amino acid sequence of human glucokinase has 31% identity with yeast hexokinase, a related enzyme for which the crystal structure has been determined. This homology has allowed us to model the three-dimensional structure of human glucokinase by analogy to the crystal structure of yeast hexokinase B. This model of human glucokinase provides a basis for understanding the effects of mutations on its enzymatic activity. Residues in the active site and on the surface of the binding cleft for glucose are highly conserved in both enzymes. Regions far from the active site are predicted to differ in conformation, and 10 insertions or deletions that range in size from 1 to 7 residues are located on the protein surface between elements of secondary structure. The model structure suggests that human glucokinase binds glucose in a similar manner to yeast hexokinase. The glucose-binding site contains a conserved aspartic acid, two conserved glutamic acids, and two conserved asparagines that form hydrogen bond interactions with the hydroxyls of the glucose similar to those observed in other sugar-binding proteins. Mutation of residues in the predicted glucose-binding site has been found to greatly reduce enzymatic activity. This model will be useful for future structure/function studies of glucokinase.

Crystallization and preliminary X-ray analysis of apo-S100 beta and S100 beta with Ca2+.

Crystals of a bovine neurotrophic factor S100 beta with and without Ca2+ bound has been crystallized by vapor diffusion from solutions of polyethylene glycol. The crystals of S100 beta with Ca2+ bound were monoclinic and belong to space group P21 with unit cell dimensions a = 36.2 A, b = 55.63 A, c = 48.39 A and beta = 111.77 degrees. The asymmetric unit contains two molecules (one dimer) which exhibit an approximate 222 point symmetry. The crystals of apo-S100 beta were tetragonal and belong to space group P4(1) with unit cell dimensions a = b = 56.04 A, c = 111.7 A. The asymmetric unit contains four molecules (two dimers) which exhibit 422 point symmetry. Both these crystal forms diffract to greater than 1.9 A resolution.

A model of the platelet factor 4 complex with heparin.

A model of heparin bound to bovine platelet factor 4 (BPF4) was completed using a graphically designed heparin molecule and the crystallographic coordinates of the native bovine platelet factor 4 tetramer. The oligosaccharides had a chain length of at least eight disaccharide units with the major repeating disaccharide unit consisting of (1----4)-O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1----4)-(2-deoxy-2-sulfamino-2-D-glucopyranosyl 6-sulfate). Each disaccharide unit carried a -4.0 charge. The structure of BPF4 was solved to 2.6 A resolution with R = 0.237. Each monomer of BPF4 contains an alpha-helix lying across 3 strands of antiparallel beta-sheet. Each helix has four lysines, which have been implicated in heparin binding. These lysine residues are predominantly on one side of the helix and are solvent accessible. Electrostatic calculations performed on the BPF4 tetramer show a ring of strong, positive charge which runs perpendicularly across the helices. Included in this ring of density is His-38, which has been shown by NMR to have a large pKa shift when heparin binds to BPF4. Our model of heparin bound to PF4 has the anionic polysaccharide perpendicular to the alpha-helices, wrapped about the tetramer along the ring of positive charge, and salt linked to all four lysines on the helix of each monomer.

Crystal structure of interleukin 8: symbiosis of NMR and crystallography.

The crystal structure of a host defense system chemotactic factor, interleukin 8, has been solved by molecular replacement using as a model the solution structure derived from nuclear magnetic resonance experiments. The structure was refined with 2 A x-ray data to an R factor of 0.187 (0.217 at 1.6 A). A comparison indicates some potential differences between the structure in solution and in the crystalline state. Our analysis also predicts that residues 4 through 9 on the amino terminus and the beta-bend, which includes His-33, may be important for receptor binding.

The three-dimensional structure of bovine platelet factor 4 at 3.0-A resolution.

Platelet factor 4 (PF4), which is released by platelets during coagulation, binds very tightly to negatively charged oligosaccharides such as heparin. To date, six other proteins are known that are homologous in sequence with PF4 but have quite different functions. The structure of a tetramer of bovine PF4 complexed with one Ni(CN)4(2-) molecule has been determined at 3.0 A resolution and refined to an R factor of 0.28. The current model contains residues 24-85, no solvent, and one overall temperature factor. Residues 1-13, which carried an oligosaccharide chain, were removed with elastase to induce crystallization; residues 14-23 and presumably 86-88 are disordered in the electron density map. Because no heavy atom derivative was isomorphous with the native crystals, the complex of PF4 with one Ni(CN)4(2-) molecule was solved using a single, highly isomorphous Pt(CN)4(2-) derivative and the iterative, single isomorphous replacement method. The secondary structure of the PF4 subunit, from amino- to carboxyl-terminal end, consists of an extended loop, three strands of antiparallel beta-sheet arranged in a Greek key, and one alpha-helix. The tetramer contains two extended, six-stranded beta-sheets, each formed by two subunits, which are arranged back-to-back to form a "beta-bilayer" structure with two buried salt bridges sandwiched in the middle. The carboxyl-terminal alpha-helices, which contain lysine residues that are thought to be intimately involved in binding heparin, are arranged as antiparallel pairs on the surface of each extended beta-sheet.

X-ray diffraction analysis of crystals of bovine platelet factor 4.

Bovine platelet factor 4 has been crystallized by "vapor dilution" in space group P2(1)2(1)2(1), a = 63.7 A, b = 66.7 A, c = 80.5 A, with four molecules, each 9505 Mr, in the asymmetric unit. The crystals diffract X-rays to better than 2.8 A resolution.