Abstract
M2 from influenza A virus functions as an oligomeric proton channel essential for the viral cycle, hence it is a high-priority pharmacological target whose structure and functions require better understanding. We studied the mechanism of M2 transmembrane domain (M2TMD) assembly in lipid membranes by the powerful biophysical technique of double electron-electron resonance (DEER) spectroscopy. By varying the M2TMD-to-lipid molar ratio over a wide range from 1:18,800 to 1:160, we found that M2TMD exists as monomers, dimers, and tetramers whose relative populations shift to tetramers with the increase of peptide-to-lipid (P/L) molar ratio. Our results strongly support the tandem mechanism of M2 assembly that is monomers-to-dimer then dimers-to-tetramer, since tight dimers are abundant at small P/L’s, and thereafter they assemble as dimers of dimers in weaker tetramers. The stepwise mechanism found for a single-pass membrane protein oligomeric assembly should contribute to the knowledge of the association steps in membrane protein folding.
Highlights
We studied the self-association of spin-labeled M2TMD21–49 at position L46C in lipid membranes of DOPC:POPS at 85:15% molar ratio
A previous study by continuous wave ESR did not report any perturbations due to spin-labeling and the results demonstrated that among several spin-labeled residues in M2, L46C follows the general pattern of structural transitions upon pH variations[44]
We studied the mechanism of assembly of M2TMD21–49, a peptide containing residues 21–49 of the M2 transmembrane domain with its N- and C-terminal juxtamembrane residues (Fig. 1b)
Summary
Analytical ultracentrifugation experiments on dodecylphosphocholine (DPC) micelle-bound M2TMD (residues 19–46) suggested monomer-tetramer equilibrium[28], which was further supported by the study of thiol-disulfide equilibrium of the same peptide in detergent[29] and lipid bilayers[15]. In the latter studies[15,29], disulfide cross-linked M2TMD dimers were generated, which assembled in tetramers. The results for detergent, β -DDM (n-Dodecyl β -D-maltoside) encompassing the 1:6,000 to 1:10 (i.e. a factor of 600) range of peptide-to-detergent molar ratios support this pathway of self-assembly, but the results are less obvious This emphasizes the significance of bilayer properties for M2 assembly and the role of stabilization of its functionally relevant forms. This study on M2TMD was enabled as a result of methodologically advancing the application of high-sensitivity DEER spectroscopy to characterize a complex equilibrium of integral membrane protein oligomers thereby contributing to our general understanding of self-association as a step in membrane protein folding
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
