title: Structural basis of Na+ activation mimicry in murine thrombin.
abstract: Unlike human thrombin, murine thrombin lacks Na+ activation due to the charge reversal substitution D222K in the Na+ binding loop. However, the enzyme is functionally stabilized in a Na+-bound form and is highly active toward physiologic substrates. The structural basis of this peculiar property is unknown. Here, we present the 2.2 A resolution x-ray crystal structure of murine thrombin in the absence of inhibitors and salts. The enzyme assumes an active conformation, with Ser-195, Glu-192, and Asp-189 oriented as in the Na+-bound fast form of human thrombin. Lys-222 completely occludes the pore of entry to the Na+ binding site and positions its side chain inside the pore, with the Nzeta atom H-bonded to the backbone oxygen atoms of Lys-185, Asp-186b, and Lys-186d. The same architecture is observed in the 1.75 A resolution structure of a thrombin chimera in which the human enzyme carries all residues defining the Na+ pore in the murine enzyme. These findings demonstrate that Na+ activation in thrombin is linked to the architecture of the Na+ pore. The molecular strategy of Na+ activation mimicry unraveled for murine thrombin is relevant to serine proteases and enzymes activated by monovalent cations in general.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University Medical School, St. Louis, Missouri 63110, USA.
authors.forename: Francesca
authors.initials: F
authors.lastname: Marino
authors.forename: Zhi-Wei
authors.initials: ZW
authors.lastname: Chen
authors.forename: Can E
authors.initials: CE
authors.lastname: Ergenekan
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Bush-Pelc
authors.forename: F Scott
authors.initials: FS
authors.lastname: Mathews
authors.forename: Enrico
authors.initials: E
authors.lastname: Di Cera
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002414:Cations, Monovalent; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D006801:Humans; D051379:Mice; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D012964:Sodium / Q000737:chemistry* / Q000378:metabolism; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D002414:Cations, Monovalent; D050506:Mutant Chimeric Proteins; D012964:Sodium; D013917:Thrombin