Viral fusion protein transmembrane domain adopts β-strand structure to facilitate membrane topological changes for virus–cell fusion

Abstract

The C-terminal transmembrane domain (TMD) of viral fusion proteins such as HIV gp41 and influenza hemagglutinin (HA) is traditionally viewed as a passive α-helical anchor of the protein to the virus envelope during its merger with the cell membrane. The conformation, dynamics, and lipid interaction of these fusion protein TMDs have so far eluded high-resolution structure characterization because of their highly hydrophobic nature. Using magicangle-spinning solid-state NMR spectroscopy, we show that the TMD of the parainfluenza virus 5 (PIV5) fusion protein adopts lipid-dependent conformations and interactions with the membrane and water. In phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, the TMD is predominantly α-helical, but in phosphatidylethanolamine (PE) membranes, the TMD changes significantly to the β-strand conformation. Measured order parameters indicate that the strand segments are immobilized and thus oligomerized. 31 P NMR spectra and small-angle X-ray scattering (SAXS) data show that this β-strand-rich conformation converts the PE membrane to a bicontinuous cubic phase, which is rich in negative Gaussian curvature that is characteristic of hemifusion intermediates and fusion pores. 1 H-31 P 2D correlation spectra and 2 H spectra show that the PE membrane with or without the TMD is much less hydrated than PC and PG membranes, suggesting that the TMD works with the natural dehydration tendency of PE to facilitate membrane merger. These results suggest a new viral-fusion model in which the TMD actively promotes membrane topological changes during fusion using the β-strand as the fusogenic conformation.solid-state NMR spectroscopy | small-angle X-ray scattering | conformational polymorphism | membrane curvature | peptide-membrane interactions V iral fusion proteins mediate entry of enveloped viruses into cells by merging the viral lipid envelope and the cell membrane. The membrane-interacting subunit of these glycoproteins contains two hydrophobic domains: a fusion peptide (FP) that is usually located at the N terminus and a transmembrane domain (TMD) at the C terminus (1). Together, these domains sandwich a water-soluble ectodomain with a helical segment that trimerizes into a coiled coil. During virus-cell fusion, the trimeric protein, which initially adopts a compact structure, unfolds to an extended intermediate that exposes the FP to the target cell membrane while keeping the TMD in the virus envelope. This extended conformation then folds onto itself to form a trimer of hairpins, in so doing pulling the cell membrane and the virus envelope into close proximity (2, 3). Subsequently, the FP and TMD are hypothesized to deform the two membranes and dehydrate them (4), eventually causing a fusion pore and a fully merged membrane. In the postfusion state, most viral fusion proteins exhibit a six-helixbundle structure in the ectodomain due to the trimer of hairpins (5).This model of virus-cell fusion largely derives from crystal structures of fusion proteins in the pre-and postfusion st...

Influenza M2 Transmembrane Domain Senses Membrane Heterogeneity and Enhances Membrane Curvature

“…This observation coincides with a recent finding that the TM domain of parainfluenza virus 5 fusion protein augments the tendency of forming curved bilayer structures 22 . TM peptide induced nonlamellar phases have also been broadly observed 62–65 .…”

Membrane curvature allosterically regulates the phosphatidylinositol cycle, controlling its rate and acyl-chain composition of its lipid intermediates

“…There were also no time-dependent changes in the spectral powder patterns. This finding of promotion of cubic phase formation has also been observed with viral fusion peptides/proteins (28,31,32), again linking cubic phases and membrane fusion.…”

“…Recently, the high-resolution structure of cubic phases has been resolved by cryo-EM tomography (26). The interface of this structure resembles intermediates that are often associated with membrane fusion phenomena (27,28). In addition, cubic phase with Pn3m symmetry has been associated with membrane fusion events (25).…”

SARS-CoV fusion peptides induce membrane surface ordering and curvature

“…Viral membrane fusion is a concerted mechanism that involves remarkable protein and lipid conformational changes. The molecular mechanism by which viral fusion proteins catalyze the membrane fusion reaction and the molecular details of lipid rearrangements in the lipid bilayer are not fully understood yet, although the current model has been constantly revisited and refined [65][66][67][68][69][70] . Crystal structures of pre-and post-fusion states of class I viral fusion proteins along with NMR structures of fusion peptides in membrane mimetics have provided a mechanistic view of the membrane fusion process.…”