Alchemical Transfer Free Energy Perturbation Simulations Reveal Entropic Mechanism Governing the Binding of a Charged Peptide to a Protein Target

Abstract We combined alchemical transfer free energy perturbation (FEP) and replica exchange with solute tempering (REST) molecular dynamics to probe the energetics of the binding of a charged peptide to a protein target. For this purpose, we have selected the coreNLS peptide KKPKKE, the conserved fragment from the nuclear localization signal (NLS) sequence in viral capsid protein binding to nuclear transporter importin-α (impα). This peptide binds to impα, natively forming a specific pose similar to that resolved experimentally. The two main outcomes of our work are as follows. First, we further develop the FEP methodology. Alchemical transfer is a rarely used version of FEP, in which an unbound peptide is gradually decoupled, while the bound peptide is coupled. Because the net charge of the system remains unchanged, no charge-related corrections within FEP are necessary. Additionally, our FEP protocol utilizes a “laissez faire” soft-walled flat-bottom spherical restraint, which does not affect a ligand as long as it occupies the major NLS binding site in impα. We showed that combining FEP with REST molecular dynamics affords exhaustive sampling of the binding process and reduces the free energy error down to 0.4 kcal/mol. Second, we found that the coreNLS binds impα with moderate affinity of ΔGb0 = −4.6 kcal/mol. We analyzed the factors contributing to binding energetics. Direct interactions between the peptide and impα favor binding and are likely to be responsible for largely native binding pose of the coreNLS. These direct binding interactions are supplemented by the gains in water–water, ion–ion, and water–ion interactions. Both, direct and indirect, enthalpic gains are about equally important, but cannot compete with severe enthalpic losses stemming from the expulsion of water and ions from the binding interface. As a result, the net binding enthalpy is unfavorable. The factor, which ultimately “saves” coreNLS binding to impα, is a strong entropic gain occurring upon water and ion release from the binding interface. Based on our investigation, we classify the mechanism governing coreNLS binding to impα as entropic. Our isothermal titration calorimetry measurements confirm the estimates of ΔGb0 provided by FEP. By connecting with our prior studies and other reports, we propose that such binding mechanism may be applicable to other charged peptides reflecting complex interplay between binding enthalpy and entropy. Interestingly, our data suggest that the ligands binding diffusively or by adopting specific poses may utilize an entropic binding mechanism.