High resolution crystal structures of free thrombin in the presence of K+ reveal the molecular basis of monovalent cation selectivity and an inactive slow form

Structural biology has recently advanced our understanding of the molecular mechanisms of activation and selectivity in monovalent cation activated enzymes. Here we report a 1.9 Å resolution crystal structure of free thrombin, a Na+ selective enzyme, in the presence of KCl. There are two molecules in the asymmetric unit, one with the cation site bound to K+ and the other with this site free. The K+-bound form shows key differences compared with the Na+-bound structure that explain the different kinetics of activation. The cation-free form, on the other hand, assumes a conformation where the monovalent cation binding site is completely disordered, the S1 pocket is inaccessible to substrate and binding to exosite I is compromised by an unprecedented > 20 Å shift in the position of the autolysis loop. This form, named S⁎, corresponds to the inactive Na+-free slow form identified by early kinetic studies. A simple model of thrombin allostery that incorporates the contribution of S⁎ is proposed.