Insights into affinity and specificity in the complexes of α-lytic protease and its inhibitorproteins: binding free energy from molecular dynamics simulation

We report the binding free energy calculation and its decomposition for the complexes of α-lytic protease and its protein inhibitors using molecular dynamics simulation. Standard mechanism serine protease inhibitors eglin C and OMTKY3 are known to have strong binding affinity for many serine proteases. Their binding loops have significant similarities, including a common P1 Leu as the main anchor in the binding interface. However, recent experiments demonstrate that the two inhibitors have vastly different affinity towards α-lytic protease (ALP), a bacterial serine protease. OMTKY3 inhibits the enzyme much more weakly (by ∼106 times) than eglin C. Moreover, a variant of OMTKY3 with five mutations, OMTKY3M, has been shown to inhibit 104 times more strongly than the wild-type inhibitor. The underlying mechanisms for the unusually large difference in binding affinities and the effect of mutation are not well understood. Here we use molecular dynamics simulation with molecular mechanics–Poisson Boltzmann/surface area method (MM-PB/SA) to investigate quantitatively the binding specificity. The calculated absolute binding free energies correctly differentiate the thermodynamic stabilities of these protein complexes, but the magnitudes of the binding affinities are systematically overestimated. Analysis of the binding free energy components provides insights into the molecular mechanism of binding specificity. The large ΔΔGbind between eglin C and wild type OMTKY3 towards ALP is mainly attributable to the stronger nonpolar interactions in the ALP-eglin C complex, arising from a higher degree of structural complementarity. Here the electrostatic interaction contributes to a lesser extent. The enhanced inhibition in the penta-mutant OMTKY3M over its wild type is entirely due to an overall improvement in the solvent-mediated electrostatic interactions in the ALP-OMTKY3M complex. The results suggest that for these protein-complexes and similar enzyme-inhibitor systems (1) the binding is driven by nonpolar interactions, opposed by overall electrostatic and solute entropy contributions; (2) binding specificity can be tuned by improving the complementarity in electrostatics between two associating proteins. Binding free energy decomposition into contributions from individual protein residues provides additional detailed information on the structural determinants and subtle conformational changes responsible for the binding specificity.