title: Conformational analysis of the full-length M2 protein of the influenza A virus using solid-state NMR.
abstract: The influenza A M2 protein forms a proton channel for virus infection and mediates virus assembly and budding. While extensive structural information is known about the transmembrane helix and an adjacent amphipathic helix, the conformation of the N-terminal ectodomain and the C-terminal cytoplasmic tail remains largely unknown. Using two-dimensional (2D) magic-angle-spinning solid-state NMR, we have investigated the secondary structure and dynamics of full-length M2 (M2FL) and found them to depend on the membrane composition. In 2D (13)C DARR correlation spectra, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-bound M2FL exhibits several peaks at β-sheet chemical shifts, which result from water-exposed extramembrane residues. In contrast, M2FL bound to cholesterol-containing membranes gives predominantly α-helical chemical shifts. Two-dimensional J-INADEQUATE spectra and variable-temperature (13)C spectra indicate that DMPC-bound M2FL is highly dynamic while the cholesterol-containing membranes significantly immobilize the protein at physiological temperature. Chemical-shift prediction for various secondary-structure models suggests that the β-strand is located at the N-terminus of the DMPC-bound protein, while the cytoplasmic domain is unstructured. This prediction is confirmed by the 2D DARR spectrum of the ectodomain-truncated M2(21-97), which no longer exhibits β-sheet chemical shifts in the DMPC-bound state. We propose that the M2 conformational change results from the influence of cholesterol, and the increased helicity of M2FL in cholesterol-rich membranes may be relevant for M2 interaction with the matrix protein M1 during virus assembly and budding. The successful determination of the β-strand location suggests that chemical-shift prediction is a promising approach for obtaining structural information of disordered proteins before resonance assignment.
authors.affiliation: Department of Chemistry, Iowa State University, Ames, Iowa, 50011.
authors.forename: Shu Yu
authors.initials: SY
authors.lastname: Liao
authors.forename: Keith J
authors.initials: KJ
authors.lastname: Fritzsching
authors.forename: Mei
authors.initials: M
authors.lastname: Hong
mesh_terms: D002784:Cholesterol / Q000378:metabolism; D002942:Circular Dichroism; D009980:Influenza A virus / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D014763:Viral Matrix Proteins / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry; D000086082:Viroporin Proteins
chemical_list: D002784:Cholesterol; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C488936:M1 protein, Influenza A virus; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins
keywords: chemical shift prediction; conformational change; influenza M2; membrane protein
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