Binding of Inhibitors to Nuclear Localization Signal Peptide from Venezuelan Equine Encephalitis Virus Capsid Protein Explored with All-Atom Replica Exchange Molecular Dynamics

Abstract

Several small molecule inhibitors have been designed to block binding of the Venezuelan equine encephalitis virus (VEEV) nuclear localization signal (NLS) sequence to the importin-α nuclear transport protein. To probe the inhibition mechanism on a molecular level, we used all-atom explicit water replica exchange molecular dynamics to study the binding of two inhibitors, I1 and I2, to the coreNLS peptide, representing the core fragment of the VEEV NLS sequence. Our objective was to evaluate the possibility of masking wherein binding of these inhibitors to the coreNLS occurs prior to its binding to importin-α. We found that the free energy of I1 and I2 binding to the coreNLS is less favorable than that to importin-α. This outcome argues against preemptive inhibitor binding to the coreNLS prior to importin-α. Instead, both inhibitors are expected to compete with the coreNLS peptide for binding to importin-α. The two factors responsible for the low affinities of the inhibitors to the coreNLS peptide are (i) the low cooperativity of binding to the peptide and (ii) the strong hydrophobic effect associated with binding to importin-α. Our results further show that upon binding to the coreNLS peptide, the inhibitors form multiple diverse binding poses. The coreNLS peptide coincubated with I1 and I2 adopts several conformational states, including open and collapsed, which underscores the fluidity of the coreNLS conformational ensemble as a target for inhibitors. Taken together with our prior investigations, this study sheds light on the molecular mechanism by which I1 and I2 ligands inhibit binding of the VEEV capsid protein to importin-α.

Figure 1

(a) Chemical structures of inhibitors G281-1564 (I1) and I2 with their structural groups identified. Differences in inhibitor structures are marked in green. (b) Structure of the coreNLS peptide KKPKKE. (c,d) Representative structures of the coreNLS peptide with bound I1 (c) and I2 (d) inhibitors. I1 primarily binds to the amino acids Lys6-Lys9, whereas I2 binds to Lys7 and Pro8 (see the text for details). Inhibitor apolar carbon and sulfur atoms are shown in light gray, whereas polar oxygen or nitrogen atoms are in red or blue. The coloring in parts (b–d) is amino acid-specific.

Figure 2

Probabilities P_b(j) of inhibitor binding to the coreNLS amino acids j. The blue and green data are collected for I1 and I2 binding. The figure shows that for all amino acids, the binding affinity of I1 is stronger than that of I2.

Figure 3

(a) Probability distributions P(RMSD_i) of the bound ligand RMSD_i values computed after aligning the coreNLS peptide structures. Data in blue and green are collected for I1 and I2 binding, respectively. The error bounds are shown by dashed lines. (b,c) Centroids of the most populated bound clusters for I1 (b) and I2 (c) inhibitors shown around the coreNLS peptide. An inhibitor pose is represented by its center of mass as a sphere. The fractions of ligand poses retained by these clusters are low, being 0.034, 0.024, 0.022, 0.019, and 0.013 for I1 and 0.038, 0.035, and 0.014 for I2 (see Section 4). The peptide structures and inhibitor poses are colored in the descending order of cluster population, as orange, yellow, cyan, purple (I1 only), and lime (I1 only). The figure implicates scattered positions of the ligands around the peptide.

Figure 4

(a) Probability distributions P(RMSD_p) of the peptides RMSD_p values computed after their alignment. (b) Probability distributions P(R_g) of the coreNLS radius of gyration R_g. In both panels, data in blue and green correspond to the peptide coincubated with I1 and I2, respectively. The data in red represent the ligand-free coreNLS peptide. The error bounds are shown by dashed lines. The figure suggests that binding of I2 but not I1 modifies the coreNLS structure.