Electrostatic interactions in hirudin-thrombin binding

Abstract

Hirudin is a good anticoagulant owing to potent inhibition of the serine protease thrombin. An aspartate- and glutamate-rich portion of hirudin plays an important part in its tight binding to thrombin through a ladder of salt bridges, and these residues have previously been mutated to asparagine or glutamine. Detailed calculations of the electrostatic contribution to changes in binding from these mutations have been performed using the finite-difference Poisson-Boltzmann method which include charge-charge interactions, solvation interactions, the residual electrostatic interaction of mutant residues, p K a shifts, and ionic strength. Single mutant effects on binding energy were close to experimental values, except for the D55N mutant whose effect is overestimated, perhaps because of displacement of a bound chloride ion from the site where it binds. Multiple mutation values were generally overestimated. The effect of p K a shifts upon the binding is significant for one hirudin residue E58, but this appears to be due to a poor salt bridge with thrombin caused by crystal contacts. Electrostatic interaction between the acidic residues is unfavorable. However, analysis of experimental multiple mutation/single mutation data shows apparently negative interactions between these residues, from which it is concluded that structural changes can occur in the complex to relieve an unfavorable interaction when more than one acidic residue is mutated. In all cases, there is a loss in stability of the complex from mutations due to loss of favorable charge-charge interactions with thrombin, but this is largely compensated for by reduced unfavorable desolvation interactions, and by residual polar interactions in the Asn/Gln mutants.