title: Cholesterol-Dependent Conformational Exchange of the C-Terminal Domain of the Influenza A M2 Protein.
abstract: The C-terminal amphipathic helix of the influenza A M2 protein plays a critical cholesterol-dependent role in viral budding. To provide atomic-level detail on the impact cholesterol has on the conformation of M2 protein, we spin-labeled sites right before and within the C-terminal amphipathic helix of the M2 protein. We studied the spin-labeled M2 proteins in membranes both with and without cholesterol. We used a multipronged site-directed spin-label electron paramagnetic resonance (SDSL-EPR) approach and collected data on line shapes, relaxation rates, accessibility of sites to the membrane, and distances between symmetry-related sites within the tetrameric protein. We demonstrate that the C-terminal amphipathic helix of M2 populates at least two conformations in POPC/POPG 4:1 bilayers. Furthermore, we show that the conformational state that becomes more populated in the presence of cholesterol is less dynamic, less membrane buried, and more tightly packed than the other state. Cholesterol-dependent changes in M2 could be attributed to the changes cholesterol induces in bilayer properties and/or direct binding of cholesterol to the protein. We propose a model consistent with all of our experimental data that suggests that the predominant conformation we observe in the presence of cholesterol is relevant for the understanding of viral budding.
authors.affiliation: Department of Chemistry and Biochemistry, Swarthmore College , Swarthmore, Pennsylvania 19081, United States.
authors.forename: Sangwoo S
authors.initials: SS
authors.lastname: Kim
authors.affiliation: Department of Chemistry and Biochemistry, Swarthmore College , Swarthmore, Pennsylvania 19081, United States.
authors.forename: Mary Alice
authors.initials: MA
authors.lastname: Upshur
authors.affiliation: Department of Chemistry and Biochemistry, Swarthmore College , Swarthmore, Pennsylvania 19081, United States.
authors.forename: Kei
authors.initials: K
authors.lastname: Saotome
authors.affiliation: Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
authors.forename: Indra D
authors.initials: ID
authors.lastname: Sahu
authors.affiliation: Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
authors.forename: Robert M
authors.initials: RM
authors.lastname: McCarrick
authors.affiliation: Department of Biophysics, National Biomedical EPR Center, Medical College of Wisconsin , Milwaukee, Wisconsin 53226, United States.
authors.forename: Jimmy B
authors.initials: JB
authors.lastname: Feix
authors.affiliation: Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
authors.forename: Gary A
authors.initials: GA
authors.lastname: Lorigan
authors.affiliation: Department of Chemistry and Biochemistry, Swarthmore College , Swarthmore, Pennsylvania 19081, United States.
authors.forename: Kathleen P
authors.initials: KP
authors.lastname: Howard
mesh_terms: D002784:Cholesterol / Q000737:chemistry*; D009980:Influenza A virus / Q000737:chemistry*; D008567:Membranes, Artificial; D008956:Models, Chemical; D010713:Phosphatidylcholines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000378:metabolism; D000086082:Viroporin Proteins
chemical_list: D002784:Cholesterol; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; C028694:1-palmitoyl-2-oleoylphosphatidylcholine