Applying Absolute Free Energy Perturbation Molecular Dynamics to Diffusively Binding Ligands.

We have developed and tested an absolute free energy perturbation (FEP) protocol, which combines all-atom molecular dynamics, replica exchange with solute tempering (REST) enhanced sampling, and a spherical harmonic restraint applied to a ligand. Our objective was to compute the binding free energy together with the underlying binding mechanism for a ligand, which binds diffusively to a protein. Such ligands represent nearly impossible targets for traditional FEP simulations. To test our FEP/REST protocol, we selected a conserved motif peptide KKPK termed minNLS from the nuclear localization signal sequence of the Venezuelan equine encephalitis virus capsid protein. This peptide fragment binds diffusively to importin-α transport protein without forming well-defined poses. Our FEP/REST simulations with a spherical restraint provided a converged estimate of minNLS binding free energy. We found that minNLS binds with moderate affinity to importin-α utilizing an unusual, purely entropic mechanism in which binding free energy is determined by favorable entropic gain. For this cationic minNLS peptide, a favorable binding entropic gain is primarily associated with the release of water from the solvation shells of charged amino acids. We demonstrated that FEP/REST simulations sample the KKPK bound ensemble well, allowing us to characterize the distribution of bound structures, binding interactions, and locations on the importin-α surface. Analysis of experimental studies offered support to our rationale behind the KKPK entropic binding mechanism.