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Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR
Matthew R Elkins1, Jonathan K Williams1, Martin D Gelenter1
1Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139.
Abstract:
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, lipid 31P 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.
Insights
Researchers identified how cholesterol interacts with the influenza M2 protein, revealing its binding site and orientation. This interaction is crucial for virus release and membrane scission, offering insights into viral budding mechanisms.
Area of Science:
- Structural Biology
- Virology
- Biophysics
Background:
- The influenza M2 protein is essential for virus budding and release, mediating membrane scission in a cholesterol-dependent manner.
- The precise atomic interactions between cholesterol and the M2 protein have remained largely unknown, hindering a full understanding of viral release mechanisms.
Purpose of the Study:
- To determine the specific cholesterol-binding site and orientation within the M2 protein in phospholipid bilayers.
- To elucidate the structural basis of cholesterol's role in M2-mediated membrane scission and viral budding.
Main Methods:
- Solid-state Nuclear Magnetic Resonance (NMR) spectroscopy was employed to investigate M2-cholesterol interactions.
- Chain-fluorinated and sterol-deuterated cholesterol analogs were used to measure cholesterol proximity and orientation relative to the M2 protein.
- Carbon-fluorine and deuterium NMR measurements provided distance and orientation restraints for structural determination.
Main Results:
- Two cholesterol molecules bind to each M2 tetramer at the C-terminal transmembrane residues, near an amphipathic helix, at 17 mol% cholesterol concentration.
- Bound cholesterol molecules are oriented parallel to the bilayer normal, with their sterol rings interacting hydrophobically with M2 transmembrane residues and polar/aromatic interactions with amphipathic helices.
- The M2-cholesterol complex induces high membrane curvature, evidenced by lipid 31P NMR spectra, facilitating membrane scission.
Conclusions:
- The determined M2-cholesterol complex structure reveals cholesterol's critical role in stabilizing M2 and inducing membrane curvature necessary for viral release.
- Cholesterol binding to M2, independent of known recognition motifs, drives M2 clustering at the budding neck, promoting membrane scission.
- The developed solid-state NMR methodology is broadly applicable for studying cholesterol-membrane protein interactions and their functional consequences.
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