Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR

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 P 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.

Entropic forces drive clustering and spatial localization of influenza A M2 during viral budding

“…The hydrocarbon rings in cholesterol confer rigidity to the molecule (30), and a connection to cholesterol-rich micro/ nanoscale structure in vivo or in vitro can therefore be established with our stiff 10e lipids on the basis of molecular flexibility in the bilayer matrix (31). While a specific interaction between M2 and cholesterol has been proposed, mediated by a putative cholesterol recognition/interaction amino acid consensus (CRAC) sequence, recent studies suggest that the actual association of cholesterol to M2 could be located in a specificity pocket between one AH and one transmembrane domain helix (28,32). Other evidence contradicts the idea M2 is associating with nanodomains (or so-called rafts) (33) and that the CRAC sequence plays a role herein (34).…”

Protein Amphipathic Helix Insertion: A Mechanism to Induce Membrane Fission

“…(3) PtdIns3P and PtdIns(3,5)P 2 for Atg18 (Gopaldass et al, 2017;Scacioc et al, 2017); (4) cholesterol for M2 protein from influenza A virus (Ekanayake et al, 2016;Elkins et al, 2017Elkins et al, , 2018Herneisen et al, 2017;Pan et al, 2019; and references therein), see Table 2.…”

NMR “Crystallography” for Uniformly (13C, 15N)‐Labeled Oriented Membrane Proteins

“…Moreover, the fluid‐like lipid environment has been shown to affect structure and function of membrane proteins . Solid‐state nuclear magnetic resonance (NMR) is a powerful spectroscopic tool which yields atomic‐level information about MPs under nearly physiological conditions . In particular, the method of oriented‐sample (OS) solid‐state NMR involves the measurements of the orientationally dependent dipolar couplings, which provide angular restraints for structure calculations.…”