Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain

Abstract

The fusion peptide (FP) and transmembrane domain (TMD) of viral fusion proteins play important roles during virus-cell membrane fusion, by inducing membrane curvature and transient dehydration. The structure of the water-soluble ectodomain of viral fusion proteins has been extensively studied crystallographically, but the structures of the FP and TMD bound to phospholipid membranes are not well understood. We recently investigated the conformations and lipid interactions of the separate FP and TMD peptides of parainfluenza virus 5 (PIV5) fusion protein F using solid-state nuclear magnetic resonance. These studies provide structural information about the two domains when they are spatially well separated in the fusion process. To investigate how these two domains are structured relative to each other in the postfusion state, when the ectodomain forms a six-helix bundle that is thought to force the FP and TMD together in the membrane, we have now expressed and purified a chimera of the FP and TMD, connected by a Gly-Lys linker, and measured the chemical shifts and interdomain contacts of the protein in several lipid membranes. The FP-TMD chimera exhibits α-helical chemical shifts in all the membranes examined and does not cause strong curvature of lamellar membranes or membranes with negative spontaneous curvature. These properties differ qualitatively from those of the separate peptides, indicating that the FP and TMD interact with each other in the lipid membrane. However, no C-C cross peaks are observed in two-dimensional correlation spectra, suggesting that the two helices are not tightly associated. These results suggest that the ectodomain six-helix bundle does not propagate into the membrane to the two hydrophobic termini. However, the loosely associated FP and TMD helices are found to generate significant negative Gaussian curvature to membranes that possess spontaneous positive curvature, consistent with the notion that the FP-TMD assembly may facilitate the transition of the membrane from hemifusion intermediates to the fusion pore.

Interplay between membrane curvature and protein conformational equilibrium investigated by solid-state NMR

“…Compared to the separate FP and TMD peptides, the FP-TMD chimera caused a relatively weak isotropic peak to DOPE (Fig. 7) (Yao et al, 2016), which is consistent with the αhelical conformation of the protein. Interestingly, addition of SDS to DOPE increased the isotropic intensity, and the isotropic peak can be assigned to a cubic phase based on 31 P T 2 relaxation times.…”

“…While studies of isolated FP and TMD peptides give structural information about the early stages of virus-cell fusion, the post-fusion state is known to involve a six-helix-bundle of the ectodomain, which should place the FP and TMD in close proximity in the membrane. We investigated the post-fusion structure of the PIV5 F protein by engineering an FP-TMD chimera, which replaces the 352-residue ectodomain of the PIV5 F protein with a short flexible linker (GGGKKKKK) (Yao et al, 2016). Interestingly, the FP-TMD chimera exhibits α-helical chemical shifts in both lamellar membranes such as POPC/POPG and negative-curvature membranes such as DOPE, without a conformational change to β-sheet (Fig.…”

“…We investigated the membrane curvature caused by the PIV5 FP and TMD using static 31 P NMR (Fig. 7) (Yang et al, 2015;Yao and Hong, 2014;Yao et al, 2016;Yao et al, 2015). These 31 P lineshapes indicate that the POPC/POPG membrane retains the lamellar structure in the presence of the FP and TMD, but the DOPE membrane shows an isotropic peak indicative of high curvature upon FP and TMD binding.…”

Insights into the mechanism of membrane fusion induced by the plant defense element, plant-specific insert

“…It is commonly accepted that the membrane fusion process involves a dehydration process. Solid-state NMR (SSNMR) is powerful in studying the topology of membrane fusion proteins (21)(22)(23)(24)(25)(26)(27). In particular, the SSNMR based H/D exchange experiments would provide insights to study the solvent accessibility of the protein, thus suggesting the level of hydration.…”

Crystal structure of the post-fusion core of theHuman coronavirus 229Espike protein at 1.86 Å resolution

“…At the same time, cleavage at an additional S2 0 site also helps to untether the fusion peptide (FP; Li, 2016;Millet & Whittaker, 2015;Belouzard et al, 2012). Following this two-step cleavage, the otherwise buried hydrophobic FP becomes exposed and available to insert into the host cell membrane (Yao et al, 2016). Subsequent conformational rearrangements within S2 result in the formation of the 3HR1-3HR2 six-helical bundle, thus bringing viral and cellular membranes into close proximity and facilitating membrane fusion (Baquero et al, 2013;Heald-Sargent & Gallagher, 2012).…”