Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR
The influenza M2 protein not only forms a proton channel but also mediates membrane scission in a cholesterol-dependent manner to cause virus budding and release. The atomic interaction of cholesterol with M2, as with most eukaryotic membrane proteins, has long been elusive. We have now determined t...
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| Format: | Article |
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National Academy of Sciences (U.S.)
2018
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| Online Access: | http://hdl.handle.net/1721.1/117075 https://orcid.org/0000-0003-4183-4979 https://orcid.org/0000-0002-7272-6885 https://orcid.org/0000-0002-6412-805X https://orcid.org/0000-0002-4581-3473 https://orcid.org/0000-0002-1567-9672 |
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| author | Sergeyev, Ivan V. Hong, Mei Elkins, Matthew Ryan Williams, Jonathan Kyle Gelenter, Martin David Dai, Peng Kwon, Byungsu Pentelute, Bradley L. |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Sergeyev, Ivan V. Hong, Mei Elkins, Matthew Ryan Williams, Jonathan Kyle Gelenter, Martin David Dai, Peng Kwon, Byungsu Pentelute, Bradley L. |
| author_sort | Sergeyev, Ivan V. |
| collection | MIT |
| description | The influenza M2 protein not only forms a proton channel but also mediates membrane scission in a cholesterol-dependent manner to cause virus budding and release. The atomic interaction of cholesterol with M2, as with most eukaryotic membrane proteins, has long been elusive. We have now determined the cholesterol-binding site of the M2 protein in phospholipid bilayers using solid-state NMR spectroscopy. Chain-fluorinated cholesterol was used to measure cholesterol proximity to M2 while sterol-deuterated cholesterol was used to measure bound-cholesterol orientation in lipid bilayers. Carbon–fluorine distance measurements show that at a cholesterol concentration of 17 mol%, two cholesterol molecules bind each M2 tetramer. Cholesterol binds the C-terminal transmembrane (TM) residues, near an amphipathic helix, without requiring a cholesterol recognition sequence motif. Deuterium NMR spectra indicate that bound cholesterol is approximately parallel to the bilayer normal, with the rough face of the sterol rings apposed to methyl-rich TM residues. The distance- and orientation-restrained cholesterol-binding site structure shows that cholesterol is stabilized by hydrophobic interactions with the TM helix and polar and aromatic interactions with neighboring amphipathic helices. At the 1:2 binding stoichiometry, lipid31P spectra show an isotropic peak indicative of high membrane curvature. This M2–cholesterol complex structure, together with previously observed M2 localization at phase boundaries, suggests that cholesterol mediates M2 clustering to the neck of the budding virus to cause the necessary curvature for membrane scission. The solid-state NMR approach developed here is generally applicable for elucidating the structural basis of cholesterol’s effects on membrane protein function. Keywords: membrane; ¹⁹F-NMR; deuterium NMR; docking; membrane scission |
| first_indexed | 2024-09-23T08:42:35Z |
| format | Article |
| id | mit-1721.1/117075 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T08:42:35Z |
| publishDate | 2018 |
| publisher | National Academy of Sciences (U.S.) |
| record_format | dspace |
| spelling | mit-1721.1/1170752022-09-23T14:01:49Z Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR Sergeyev, Ivan V. Hong, Mei Elkins, Matthew Ryan Williams, Jonathan Kyle Gelenter, Martin David Dai, Peng Kwon, Byungsu Pentelute, Bradley L. Massachusetts Institute of Technology. Department of Chemistry Elkins, Matthew Ryan Williams, Jonathan Kyle Gelenter, Martin David Dai, Peng Kwon, Byungsu Pentelute, Bradley L. The influenza M2 protein not only forms a proton channel but also mediates membrane scission in a cholesterol-dependent manner to cause virus budding and release. The atomic interaction of cholesterol with M2, as with most eukaryotic membrane proteins, has long been elusive. We have now determined the cholesterol-binding site of the M2 protein in phospholipid bilayers using solid-state NMR spectroscopy. Chain-fluorinated cholesterol was used to measure cholesterol proximity to M2 while sterol-deuterated cholesterol was used to measure bound-cholesterol orientation in lipid bilayers. Carbon–fluorine distance measurements show that at a cholesterol concentration of 17 mol%, two cholesterol molecules bind each M2 tetramer. Cholesterol binds the C-terminal transmembrane (TM) residues, near an amphipathic helix, without requiring a cholesterol recognition sequence motif. Deuterium NMR spectra indicate that bound cholesterol is approximately parallel to the bilayer normal, with the rough face of the sterol rings apposed to methyl-rich TM residues. The distance- and orientation-restrained cholesterol-binding site structure shows that cholesterol is stabilized by hydrophobic interactions with the TM helix and polar and aromatic interactions with neighboring amphipathic helices. At the 1:2 binding stoichiometry, lipid31P spectra show an isotropic peak indicative of high membrane curvature. This M2–cholesterol complex structure, together with previously observed M2 localization at phase boundaries, suggests that cholesterol mediates M2 clustering to the neck of the budding virus to cause the necessary curvature for membrane scission. The solid-state NMR approach developed here is generally applicable for elucidating the structural basis of cholesterol’s effects on membrane protein function. Keywords: membrane; ¹⁹F-NMR; deuterium NMR; docking; membrane scission 2018-07-24T15:36:04Z 2018-07-24T15:36:04Z 2017-11 2017-08 2018-07-18T18:13:05Z Article http://purl.org/eprint/type/JournalArticle 0027-8424 1091-6490 http://hdl.handle.net/1721.1/117075 Elkins, Matthew R. et al. “Cholesterol-Binding Site of the Influenza M2 Protein in Lipid Bilayers from Solid-State NMR.” Proceedings of the National Academy of Sciences 114, 49 (November 2017): 12946–12951 © 2017 National Academy of Sciences https://orcid.org/0000-0003-4183-4979 https://orcid.org/0000-0002-7272-6885 https://orcid.org/0000-0002-6412-805X https://orcid.org/0000-0002-4581-3473 https://orcid.org/0000-0002-1567-9672 http://dx.doi.org/10.1073/PNAS.1715127114 Proceedings of the National Academy of Sciences Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf National Academy of Sciences (U.S.) PNAS |
| spellingShingle | Sergeyev, Ivan V. Hong, Mei Elkins, Matthew Ryan Williams, Jonathan Kyle Gelenter, Martin David Dai, Peng Kwon, Byungsu Pentelute, Bradley L. Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR |
| title | Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR |
| title_full | Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR |
| title_fullStr | Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR |
| title_full_unstemmed | Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR |
| title_short | Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR |
| title_sort | cholesterol binding site of the influenza m2 protein in lipid bilayers from solid state nmr |
| url | http://hdl.handle.net/1721.1/117075 https://orcid.org/0000-0003-4183-4979 https://orcid.org/0000-0002-7272-6885 https://orcid.org/0000-0002-6412-805X https://orcid.org/0000-0002-4581-3473 https://orcid.org/0000-0002-1567-9672 |
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