Probing the metastable state of influenza hemagglutinin

Abstract

Viral entry into host cells is mediated by membrane proteins in a metastable state that transition to a more stable state upon a stimulus. For example, in the influenza envelope protein hemagglutinin (HA), the low pH in the endosome triggers a transition from the metastable prefusion conformation to the stable fusion conformation. To identify probes that interfere with HA function, here we screened a library of H7 HA peptides for inhibition of H7 HA-mediated entry. We discovered a peptide, PEP87 (WSYNAELLVAMENQHTI), that inhibited H7 and H5 HA-mediated entry. PEP87 corresponds to a highly conserved helical region of the HA2 subunit of HA that self-interacts in the neutral pH conformation. Mutagenesis experiments indicated that PEP87 binds to its native region in the HA trimer. We also found that PEP87 is unstructured in isolation but tends to form a helix as evidenced by CD and NMR studies. Fluorescence, chemical cross-linking, and saturation transfer difference NMR data suggested that PEP87 binds to the neutral pH conformation of HA and disrupts the HA structure without affecting its oligomerization state. Together, this work provides support for a model in which PEP87 disrupts HA function by displacing native interactions of the neutral pH conformation. Moreover, our observations indicate that the HA prefusion structure (and perhaps the metastable states of other viral entry proteins) is more dynamic with transient motions being larger than generally appreciated. These findings also suggest that the ensemble of prefusion structures presents many potential sites for targeting in therapeutic interventions.

pH-Dependent Mechanisms of Influenza Infection Mediated by Hemagglutinin

“…Fourth, MS H/D exchange increases at intermediate pH suggesting increased exposure of exchangeable groups and dynamics/flexibility (Garcia et al, 2015). Fifth, the binding of an inhibitor peptide to a nonexposed region of HA suggests the presence of large breathing motions (Kingsley et al, 2017). Sixth, molecular dynamics studies of HA suggest the presence of intermediate conformations and larger flexibility at low pH (Lin et al, 2016;Burke et al, 2020).…”

Targeting Hemagglutinin: Approaches for Broad Protection against the Influenza A Virus

“…Compounds such as CL-61917, CL-385319, and CL-62554 [208], BMY-27709 [209], LY-180299 [210], RO5464466 and RO5487624 [211], FA-583 and FA-617 [212] target group I HAs, whereas TBHQ [10,213], S19 and C22 [214] are fusion inhibitors to group II HAs. Recently, peptides such as PEP87 were found to be able to bind to the HA trimmer and thus disrupted HA-mediated entry [215]. Arbidol was found to inhibit HA for both group I and group II HAs by binding to a hydrophobic cavity in the stem region [216,217].…”