Crystallization and Preliminary X-ray Diffraction Analysis of Two Conformations of Intact Human Antithrombin

Abstract

Human antithrombin has been crystallized by microdialysis at pH 6·7 using 18% (w/v) polyethylene glycol-4000 as precipitant. Under these conditions two crystal forms grew. The first started growing after ten days, diffracted to 3·0 Å resolution and belongs to the monoclinic space group P 2 1 with two molecules in the asymmetric unit and unit cell dimensions a=70·1 Å, b=101·5 Å, c =90·5 Å and β=105·9 Å. The other crystal form took more than three months to appear, diffracted to 5·5 Å and belongs to the hexagonal space group of either P6 1 or P 6 5 with unit cell dimensions of a = b=99·3 Å and c= 152·9 Å and two molecules in the asymmetric unit. The antithrombin redissolved from the monoclinic crystals was shown both by SDS-polyacrylamide gel electrophoresis and by protein sequence analysis to be intact while that from the hexagonal crystals was cleaved in the reactive centre loop between the P′2 and P′3 (i.e. Leu-Asn) residues. Further analysis of the intact inhihitor from the monoclinic crystals indicated that the antithrombin was present in two different conformations; an active form which could inhibit thrombin and form a stable complex with the protease, and a form which was inactive as an inhibitor and which also did not act as a substrate for thrombin. This latter form also had a low affinity for heparin and in these ways resembles latent antithrombin. The active material from the monoclinic crystals had an association rate constant with thrombin in the presence of heparin ( k ass) of 7·5 × 10 7 M -1 s -1 ( k ass for native antithrombin = 8·2 (± 1·0) × 10 7 M -1 s -1) indicating it still had effective heparin cofactor activity. X-ray diffraction analysis also suggests that two different protein conformations exist within the monoclinic crystals. Whereas the rotation function peak heights are equal for both molecules in the asymmetric unit using the structure of intact ovalbumin as a search model, one of the two molecules gives a much clearer signal than the other when the structures of the two cleaved serpins, α1-antitrypsin and α1-antichymotrypsin are used.