Loop electrostatics asymmetry modulates the pre-existing conformational equilibrium in thrombin

By N. Pozzi, M. Zerbetto, L. Acquasaliente, S. Tescari, D. Frezzato, A. Polimeno, D. Gohara, E. Di Cera, V. De Filippis. 2016

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Thrombin exists as an ensemble of active (E) and inactive (E*) conformations that differ for 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.

Subfields: Hematology, Molecular Biology