The isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin: Changes accompanying activation and exosite binding to thrombin

摘要: The X-ray crystal structure of prethrombin2 (pre2), the immediate inactive precursor alpha-thrombin, has been determined at 2.0 A resolution complexed with hirugen. refined to a final R-value 0.169 using 14,211 observed reflections in range 8.0-2.0 A. total 202 water molecules have also located structure. Comparison hirugen-thrombin complex showed that, apart from flexible beginning and terminal regions molecule, there are 4 polypeptide segments pre2 differing conformation active enzyme (Pro 186-Asp 194, Gly 216-Gly 223, 142-Pro 152, Arg 15-Ile 16 cleavage region). formation Ile 16-Asp 194 ion pair specificity pocket characteristic serine protease activation catalytic triad being conserved. With determination isomorphous structures D-Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, changes that occur site affect kinetics chromogenic substrate hydrolysis on binding fibrinogen recognition exosite determined. backbone Ala 190-Gly 197 segment an average RMS difference 0.55 between 2 (about 3.7 sigma compared bulk structure). This type II beta-bends, first bend showing largest shift due hirugen binding. Another important feature was different conformations side chain Glu 192. extends solvent hirugen-thrombin, which is compatible substrates having acidic residue P3 position (protein-C, thrombin platelet receptor). In PPACK-thrombin, Asp 189 221A-Gly 223 move provide space for inhibitor, whereas movement expands region. Although 8 expelled PPACK binding, inhibitor resolvated 5 other molecules.