Side-chain conformation of the M2 transmembrane peptide proton channel of influenza a virus from 19F solid-state NMR.

The M2 transmembrane peptide (M2TMP) of the influenza A virus forms a tetrameric helical bundle that acts as a proton-selective channel important in the viral life cycle. The side-chain conformation of the peptide is largely unknown and is important for elucidating the proton-conducting mechanism and the channel stability. Using a 19 F spin diffusion NMR technique called CODEX, we have measured the oligomeric states and interhelical side chain-side chain 19 F- 19 F distances at several residues using singly fluorinated M2TMP bound to DMPC bilayers. 19 F CODEX data at a key residue of the proton channel, Trp 41 , confirm the tetrameric state of the peptide and yield a nearest-neighbor interhelical distance of ∼11 A under both neutral and acidic pH. Since the helix orientation is precisely known from previous 15 N NMR experiments and the backbone channel diameter has a narrow allowed range, this 19 F distance constrains the Trp 41 side-chain conformation to t90 (Χ 1 ≈ 180°, Χ 2 ≈ 90°). This Trp 41 rotamer, combined with a previously measured 15 N- 13 C distance between His 37 and Trp 41 1 , suggests that the His 37 rotamer is t-160. The implication of the proposed (His 37 , Trp 41 ) rotamers to the gating mechanism of the M2 proton channel is discussed. Binding of the antiviral drug amantadine to the peptide does not affect the F-F distance at Trp 41 . Interhelical 19 F- 19 F distances are also measured at residues 27 and 38, each mutated to 4- 19 F-Phe. For V27F-M2TMP, the 19 F- 19 F distances suggest a mixture of dimers and tetramers, whereas the L38F-M2TMP data indicate two tetramers of different sizes, suggesting side chain conformational heterogeneity at this lipid-facing residue. This work shows that 19 F spin diffusion NMR is a valuable tool for determining long-range intermolecular distances that shed light on the mechanism of action and conformational heterogeneity of membrane protein oligomers.