Competitive Binding of Viral Nuclear Localization Signal Peptide and Inhibitor Ligands to Importin‑α Nuclear Transport Protein

• Published in: Journal of chemical information and modeling 2024-07, Vol.64 (13), p.5262-5272
• Main Authors: Delfing, Bryan M., Laracuente, Xavier E., Jeffries, William, Luo, Xingyu, Olson, Audrey, Foreman, Kenneth W., Petruncio, Greg, Lee, Kyung Hyeon, Paige, Mikell, Kehn-Hall, Kylene, Lockhart, Christopher, Klimov, Dmitri K.
• Format: Article
• Language: English
• Subjects: alpha Karyopherins - antagonists & inhibitors; alpha Karyopherins - chemistry; alpha Karyopherins - metabolism; Amino Acid Sequence; Amino acids; Binding sites; Binding, Competitive; Competition; Computational Biochemistry; Encephalitis; Encephalitis Virus, Venezuelan Equine - chemistry; Encephalitis Virus, Venezuelan Equine - metabolism; Free energy; Inhibitors; Ligands; Localization; Molecular dynamics; Molecular Dynamics Simulation; Nuclear Localization Signals - chemistry; Peptides; Peptides - chemistry; Peptides - metabolism; Peptides - pharmacology; Protein Binding; Proteins; Simulation; Structural analysis
• ISSN: 1549-9596; 1549-960X; 1549-960X
• DOI: 10.1021/acs.jcim.4c00626

Venezuelan equine encephalitis virus (VEEV) is a highly virulent pathogen whose nuclear localization signal (NLS) sequence from capsid protein binds to the host importin-α transport protein and blocks nuclear import. We studied the molecular mechanisms by which two small ligands, termed I1 and I2, interfere with the binding of VEEV’s NLS peptide to importin-α protein. To this end, we performed all-atom replica exchange molecular dynamics simulations probing the competitive binding of the VEEV coreNLS peptide and I1 or I2 ligand to the importin-α major NLS binding site. As a reference, we used our previous simulations, which examined noncompetitive binding of the coreNLS peptide or the inhibitors to importin-α. We found that both inhibitors completely abrogate the native binding of the coreNLS peptide, forcing it to adopt a manifold of nonnative loosely bound poses within the importin-α major NLS binding site. Both inhibitors primarily destabilize the native coreNLS binding by masking its amino acids rather than competing with it for binding to importin-α. Because I2, in contrast to I1, binds off-site localizing on the edge of the major NLS binding site, it inhibits fewer coreNLS native binding interactions than I1. Structural analysis is supported by computations of the free energies of the coreNLS peptide binding to importin-α with or without competition from the inhibitors. Specifically, both inhibitors reduce the free energy gain from coreNLS binding, with I1 causing significantly larger loss than I2. To test our simulations, we performed AlphaScreen experiments measuring IC50 values for both inhibitors. Consistent with in silico results, the IC50 value for I1 was found to be lower than that for I2. We hypothesize that the inhibitory action of I1 and I2 ligands might be specific to the NLS from VEEV’s capsid protein.