Solvent accessibility of the thrombin-thrombomodulin interface

The kinetics of solvent accessibility at the protein-protein interface betw een thrombin and a fragment of thrombomodulin, TMEGF45, have been monitored by amide hydrogen/deuterium (H/H-2) exchange detected by MALDI-TOF mass sp ectrometry. The interaction is rapid and reversible, requiring development of theory and experimental methods to distinguish H/H-2 exchange due to sol vent accessibility at the interface from H/H-2 exchange due to complex diss ociation. Association and dissociation rate constants were measured by surf ace plasmon resonance and amide H/H-2 exchange rates were measured at diffe rent pH values and concentrations of TMEGF45. When essentially 100 % of the thrombin was bound to TMEGF45, two segments of thrombin became completely solvent-inaccessible, as evidenced by the pH insensitivity of the amide H/H -2 exchange rates. These segments form part of anion-binding exosite I and contain the residues for which alanine substitution abolishes TM binding. S everal other regions of thrombin showed slowing of amide exchange upon TMEG F45 binding, but the exchange remained pH-dependent, suggesting that these regions of thrombin were rendered only partially solvent-inaccessible by TM EGF45 binding. These partially inaccessible regions of thrombin form both s urface and buried contacts into the active site of thrombin and contain res idues implicated in allosteric changes in thrombin upon TM binding. (C) 200 1 Academic Press.