title: Neutron and X-ray crystallographic analysis of the human α-thrombin-bivalirudin complex at pD 5.0: protonation states and hydration structure of the enzyme-product complex.
abstract: The protonation states and hydration structures of the α-thrombin-bivalirudin complex were studied by joint XN refinement of the single crystal X-ray and neutron diffraction data at resolutions of 1.6 and 2.8Å, respectively. The atomic distances were estimated by carrying out X-ray crystallographic analysis at 1.25Å resolution. The complex represents a model of the enzyme-product (EP) complex of α-thrombin. The neutron scattering length maps around the active site suggest that the side chain of H57/H was deuterated. The joint XN refinement showed that occupancies for Dδ1 and Dε2 of H57/H were 1.0 and 0.7, respectively. However, no significant neutron scattering length density was observed around the hydroxyl oxygen Oγ of S195/H, which was close to the carboxylic carbon atom of dFPR-COOH. These observations suggest that the Oγ atom of S195/H is deprotonated and maintains its nucleophilicity in the EP complex. In addition to the active site, the hydration structures of the S1 subsite and the Exosite I, which are involved in the recognition of bivalirudin, are presented.
authors.affiliation: Ibaraki University, Tokai, Ibaraki, Japan. taro@mx.ibaraki.ac.jp
authors.forename: Taro
authors.initials: T
authors.lastname: Yamada
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kurihara
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ohnishi
authors.forename: Taro
authors.initials: T
authors.lastname: Tamada
authors.forename: Katsuaki
authors.initials: K
authors.lastname: Tomoyori
authors.forename: Kenji
authors.initials: K
authors.lastname: Masumi
authors.forename: Ichiro
authors.initials: I
authors.lastname: Tanaka
authors.forename: Ryota
authors.initials: R
authors.lastname: Kuroki
authors.forename: Nobuo
authors.initials: N
authors.lastname: Niimura
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006629:Hirudins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D033363:Neutron Diffraction; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism; D014867:Water / Q000737:chemistry*
chemical_list: D006629:Hirudins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014867:Water; D013917:Thrombin; C074619:bivalirudin