Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin

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