The Transmembrane Conformation of the Influenza B Virus M2 Protein in Lipid Bilayers

Abstract

Influenza A and B viruses cause seasonal flu epidemics. The M2 protein of influenza B (BM2) is a membrane-embedded tetrameric proton channel that is essential for the viral lifecycle. BM2 is a functional analog of AM2 but shares only 24% sequence identity for the transmembrane (TM) domain. The structure and function of AM2, which is targeted by two antiviral drugs, have been well characterized. In comparison, much less is known about the structure of BM2 and no drug is so far available to inhibit this protein. Here we use solid-state NMR spectroscopy to investigate the conformation of BM2(1–51) in phospholipid bilayers at high pH, which corresponds to the closed state of the channel. Using 2D and 3D correlation NMR experiments, we resolved and assigned the 13 C and 15 N chemical shifts of 29 residues of the TM domain, which yielded backbone (φ, ψ) torsion angles. Residues 6–28 form a well-ordered α-helix, whereas residues 1–5 and 29–35 display chemical shifts that are indicative of random coil or β-sheet conformations. The length of the BM2-TM helix resembles that of AM2-TM, despite their markedly different amino acid sequences. In comparison, large 15 N chemical shift differences are observed between bilayer-bound BM2 and micelle-bound BM2, indicating that the TM helix conformation and the backbone hydrogen bonding in lipid bilayers differ from the micelle-bound conformation. Moreover, H N chemical shifts of micelle-bound BM2 lack the periodic trend expected for coiled coil helices, which disagree with the presence of a coiled coil structure in micelles. These results establish the basis for determining the full three-dimensional structure of the tetrameric BM2 to elucidate its proton-conduction mechanism.

Atomic structures of closed and open influenza B M2 proton channel reveal the conduction mechanism

“…5 ) show very similar chemical shifts for the TM residues in the POPE and POPC : POPG membranes, indicating that the TM helix conformation is unaffected by the membrane curvature. This observation is consistent with the invariance of BM2 chemical shifts between DLPE and POPC : POPG membranes at high pH 29 . Because the cytoplasmic domain forms a β-strand at low pH but is disordered at high pH, the membrane curvature is correlated with the β-strand conformation.…”

“…1b – c , Extended Data Figs. 2 and 3 ), following previous assignment of a similar construct 29 . We utilized sequential and medium-range cross peaks ( i to i +1 or i +2) in the 3D NCCX and 3D CCC spectra to facilitate assignment of overlapping peaks.…”

Pulsed Third-Spin-Assisted Recoupling NMR for Obtaining Long-Range13C-13C and15N-13C Distance Restraints

“…Microcrystalline GB1 samples were expressed and purified as previously described [35][36] . Glucagon peptide was purchased from Biopeptek Pharmaceuticals (Malvern, PA) and fibrillized in pH 2 solution at 21˚C for 7 days at a concentration of 8 mg/ml 37 .…”

Challenges and Perspectives in Chemical Synthesis of Highly Hydrophobic Peptides

“…Several studies demonstrated that transmembrane proteins spontaneously fold while incorporated in an artificial lipid membrane. This behavior is utilized in analytical applications such as circular dichroism (CD) spectroscopy and solution nuclear magnetic resonance (NMR) studies (Engelman and Steitz, 1981;Ladokhin et al, 2010;Mandala et al, 2019). In order to aid NCL multiple surfactants were investigated to increase the solubility of transmembrane peptides in guanidinium chloride or urea as additives to ligation buffer.…”