Binding of Inhibitors\nto Nuclear Localization Signal\nPeptide from Venezuelan Equine Encephalitis Virus Capsid Protein Explored\nwith All-Atom Replica Exchange Molecular Dynamics

Several small molecule inhibitors have been designed\nto block binding\nof the Venezuelan equine encephalitis virus (VEEV) nuclear localization\nsignal (NLS) sequence to the importin-α nuclear transport protein.\nTo probe the inhibition mechanism on a molecular level, we used all-atom\nexplicit water replica exchange molecular dynamics to study the binding\nof two inhibitors, I1 and I2, to the coreNLS peptide, representing\nthe core fragment of the VEEV NLS sequence. Our objective was to evaluate\nthe possibility of masking wherein binding of these inhibitors to\nthe coreNLS occurs prior to its binding to importin-α. We found\nthat the free energy of I1 and I2 binding to the coreNLS is less favorable\nthan that to importin-α. This outcome argues against preemptive\ninhibitor binding to the coreNLS prior to importin-α. Instead,\nboth inhibitors are expected to compete with the coreNLS peptide for\nbinding to importin-α. The two factors responsible for the low\naffinities of the inhibitors to the coreNLS peptide are (i) the low\ncooperativity of binding to the peptide and (ii) the strong hydrophobic\neffect associated with binding to importin-α. Our results further\nshow that upon binding to the coreNLS peptide, the inhibitors form\nmultiple diverse binding poses. The coreNLS peptide coincubated with\nI1 and I2 adopts several conformational states, including open and\ncollapsed, which underscores the fluidity of the coreNLS conformational\nensemble as a target for inhibitors. Taken together with our prior\ninvestigations, this study sheds light on the molecular mechanism\nby which I1 and I2 ligands inhibit binding of the VEEV capsid protein\nto importin-α.