Crystal structure of bovine antithrombin III

Abstract

The crystal structure of bovine antithrombin III has been solved by molecular replacement, using a~-antitrypsin (30% sequence homology with human antithrombin III) as a model. The protein crystallizes with two molecules in the asymmetric unit. The use of different resolution ranges was essential in determining the true orientations of the two molecules, because these were the orientations that appeared most consistently in the rotation function, albeit with different scores and slightly different values. Accuracy and correctness of the orientations of the two independent molecules was crucial for the success of the translation function. Stripping off surface atoms from the trial model resulted in a better signal-to-noise ratio in the Crowther-Blow translation function, which turned out to be very discriminative, even though the translational search was performed with less than 30% of the asymmetric unit. In this way, the orientation and position of each of the two independent molecules was separately determined; the crystal packing of the corresponding dimer showed no bad contacts. Phases calculated with this model allowed the determination of heavy-atom sites in a derivative which had previously resisted interpretation. The current R factor after energy minimization and a l ps moleculardynamics simulation is 32% at 3.6 ,~ resolution. In~oducfionAntithrombin III (Mr = 56 600) is a plasma antiserine protease involved in the coagulation process. Its physiological role is thought to be the inhibition of thrombin and/or factor Xa; factor Xa, together with factor Va and Ca 2÷ ions, helps cleave prothrombin into thrombin, which then cleaves fibrinogen into fibrin (Furie & Furie, 1988). Fibrin then polymerizes in molecular nets which, together with activated platelets, repair blood vessel damage around a wound. The rate of inhibition by antithrombin III (ATIII) is modulated by a polysaccharide, heparin (Rosenberg & Damus, 1973 (Blackburn, Smith & Sibley, 1983) and genetic studies (Chang & Tran, 1986), and is located in the N-terminal domain of the protein. Furthermore, a pentasaccharide that presents the same activity as heparin for the inactivation of factor Xa by antithrombin has been chemically synthesized (Choay, Petitou, Lormeau, Sinay, Casu & Gatti, 1983;Sinay et al., 1984). However, specific contacts at the molecular level between heparin and ATIII are unknown and this prevents any rational design of chemically synthesized analogs of heparin. In order to make such drug-design experiments and molecular modeling possible, the three-dimensional structure of ATIII has to be known. At a later stage, the complex between the pentasaccharide and ATIII will also have to be solved.In this article, we report the crystal structure determination of bovine ATIII. Our strategy was to pursue isomorphous replacement and molecular replacement methods separately. The latter method proved to be successful and helped interpret some results from the first. Because of some difficulties encountered in the application of the ...

On the use of low-frequency normal modes to enforce collective movements in refining macromolecular structural models

“…Highly partial models such as C-alphas-only models can be used and refined in MR techniques, as documented elsewhere (38,39). The NM refinement program described here was successfully used to discriminate between alternative solutions of the MR problem, in a manner reminiscent of the Patterson-correlation-refinement method (40).…”

The Contribution of the Conserved Hinge Region Residues of .alpha.1-Antitrypsin to Its Reaction with Elastase

“…The first of these is the serpin prior to contact with the enzyme, which probably has a five-stranded P-sheet A and a helical reactive-site loop as seen in the ovalbumin crystal structure (Stein et al, 1990) or the al-antichymotrypsin crystal structure (Wei et al, 1994). The second structure is the well-known structure of the reactive-site loopcleaved serpin with the hinge region and reactive-site loop incorporated into the A-sheet (Baumann et al, 1991;Delarue et al, 1990;Lobermann et al, 1984). Plasminogen activator inhibitor-1 and antithrombin show a third structure (Carrell et al, 1994;Mottonen et al, 1992), where dissociation of strand 1C from the C-sheet has permitted the reactive-site loop and hinge region to be fully incorporated into the A-sheet without cleavage of the reactive-site loop.…”

“…If it is assumed that an S to R-like transition and insertion of the hinge region into the A /3-sheet are essential for the formation of the stable serpin-enzyme complex (El*), the effect of blocking this transition would be to favor proteolytic cleavage of the reactive-site loop (i.e., formation of T). Examination of the crystal structure of a.;-AT (Lobermann et al, 1984) and other serpins (Carrell et al, 1994;Delarue et al, 1990;Mottonen et al, 1992) indicates that substitution of larger or charged amino acids in the hinge region would hinder, or raise the energy barrier for, strand insertion. Previous data (Hood et al, 1994;Lawrence et al, 1994;Schulze et al, 1991;Skriver et al, 1991) have clearly demonstrated that mutagenesis of the P14 and P12 residues, especially to charged amino acids, interferes with normal inhibition behavior.…”

The molecular structure of the complex of Ascaris chymotrypsin/elastase inhibitor with porcine elastase

“…Detailed thermodynamic association constants for ovomucoid inhibitor variants with several serine proteinases have been determined [9]. Crystal structures have been reported for bovine pancreatic trypsin inhibitor (Kunitz-Kunin family; [10,11]), avian ovomucoid inhibitor third domain (Kazal family; [12][13][14][15][16]), Streptomycessubtilisin inhibitor (SSI family; [17]), soybean trypsin inhibitor and Erythina trypsin inhibitor (STI-Kunitz family; [18,19]), barley seed chymotrypsin inhibitor 2 [20,21] and leech inhibitor eglin-c (PI-1 family; [22][23][24][25]), polypeptide chymotrypsin inhibitor from potato (PI-2 family; [26]), acid-stable inhibitor from human mucus secretions (chelonianin family; [27]), Bowman-Birk inhibitors [28][29][30], squash seed trypsin inhibitor (squash seed inhibitor family; [31]), serpins [32][33][34][35][36][37][38] and hirudin [39,40]. The inhibitors from these different families have a common conformation for the reactive-site loop, while displaying completely different overall structures (see [41,42] for reviews).…”