Structure and Mechanisms of Actions of Curvature-Inducing Viral Membrane Proteins from Solid-State NMR

Abstract

Many membrane proteins such as viral fusion proteins induce membrane curvature for function. To understand how protein structure and membrane properties underlie such curvature induction, we have applied and developed solid-state NMR spectroscopic techniques. The conformation, topology, and lipid interactions of the fusion peptide (FP) and transmembrane domain (TMD) of a paramyxovirus fusion protein are investigated. 13C and 15N chemical shifts indicate that both the FP and TMD adopt lipid-dependent conformations, with the α-helical conformation dominant in low-curvature phosphocholine and phosphoglycerol membranes while the β-strand conformation dominant in negative-curvature phosphoethanolamine (PE) membranes. 31P and 2H NMR spectra, together with small-angle X-ray scattering results, indicate that the PE membrane is transformed by the TMD into a bicontinuous cubic phase, which is rich in negative Gaussian curvature, the type of curvature in hemifusion intermediates and fusion pores. These results strongly suggest that the two hydrophobic termini of this viral fusion protein actively facilitate membrane topological changes to promote membrane merger, and the β-strand is the fusogenic conformation.To detect membrane curvature and the binding site of proteins in mixed-curvature phospholipid membranes, we have developed a solid-state NMR approach based on magnetically oriented bicelles, relaxation NMR and 2D correlation experiments under off-magic-angle spinning (OMAS). We demonstrate this approach on the influenza M2 protein, which mediates virus budding by membrane scission. An M2 peptide encompassing the TMD and an amphipathic helix in the cytoplasmic domain is compared with the TMD alone. Our results indicate that the amphipathic helix causes high membrane curvature, moreover, the peptide preferentially partitions into this high-curvature domain. This bicelle-based solid-state NMR approach is generally applicable to curvature-inducing membrane proteins.