title: Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin.
abstract: Thrombin exists as an ensemble of active (E) and inactive (E*) conformations that differ in their accessibility to the active site. Here we show that redistribution of the E*-E equilibrium can be achieved by perturbing the electrostatic properties of the enzyme. Removal of the negative charge of the catalytic Asp102 or Asp189 in the primary specificity site destabilizes the E form and causes a shift in the 215-217 segment that compromises substrate entrance. Solution studies and existing structures of D102N document stabilization of the E* form. A new high-resolution structure of D189A also reveals the mutant in the collapsed E* form. These findings establish a new paradigm for the control of the E*-E equilibrium in the trypsin fold.
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine , St. Louis, Missouri 63104, United States.
authors.forename: Nicola
authors.initials: N
authors.lastname: Pozzi
authors.forename: Mirco
authors.initials: M
authors.lastname: Zerbetto
authors.forename: Laura
authors.initials: L
authors.lastname: Acquasaliente
authors.forename: Simone
authors.initials: S
authors.lastname: Tescari
authors.forename: Diego
authors.initials: D
authors.lastname: Frezzato
authors.forename: Antonino
authors.initials: A
authors.lastname: Polimeno
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine , St. Louis, Missouri 63104, United States.
authors.forename: David W
authors.initials: DW
authors.lastname: Gohara
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine , St. Louis, Missouri 63104, United States.
authors.forename: Enrico
authors.initials: E
authors.lastname: Di Cera
authors.forename: Vincenzo
authors.initials: V
authors.lastname: De Filippis
mesh_terms: D055162:Biocatalysis; D018360:Crystallography, X-Ray; D006801:Humans; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D055672:Static Electricity; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D013917:Thrombin