Side-Chain Conformation of the M2 Transmembrane Peptide Proton Channel of Influenza A Virus from ¹⁹F Solid-State NMR

Abstract

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 19F spin diffusion NMR technique called CODEX, we have measured the oligomeric states and interhelical side chain-side chain 19F-19F distances at several residues using singly fluorinated M2TMP bound to DMPC bilayers. 19F CODEX data at a key residue of the proton channel, Trp41, confirm the tetrameric state of the peptide and yield a nearest-neighbor interhelical distance of approximately 11 A under both neutral and acidic pH. Since the helix orientation is precisely known from previous 15N NMR experiments and the backbone channel diameter has a narrow allowed range, this 19F distance constrains the Trp41 side-chain conformation to t90 (chi1 approximately 180 degrees , chi2 approximately 90 degrees ). This Trp41 rotamer, combined with a previously measured 15N-13C distance between His37 and Trp411, suggests that the His37 rotamer is t-160. The implication of the proposed (His37, Trp41) 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 Trp41. Interhelical 19F-19F distances are also measured at residues 27 and 38, each mutated to 4-19F-Phe. For V27F-M2TMP, the 19F-19F 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 19F 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.

Acid activation mechanism of the influenza A M2 proton channel

“…Moreover, one of the Trp41 residues flipped to the t90 conformer, as found in the crystal structures (9,24,55), during this simulation. This is consistent with another very long simulation study in which all four Trp41 residues eventually flipped to the t90 conformation (32), experimental support of the t90 conformation (9,12,24,55,56), and our own PMF calculation of the Trp41 χ 2 dihedral angle rotation discussed earlier in this paper. Therefore, to facilitate the equilibration, the remainder of the Trp41 side chains were manually rotated from the t-105 rotamer to the t90 rotamer.…”

“…S3) (the equilibration starting from the t-105 rotamer of Trp41 because in the NMR structure 2L0J does not yield a stable protein structure within 200-ns simulation). Our decision to change to the t90 rotamer of Trp41 is supported by crystal structures (9,24,55), SSNMR measurements (12,56), as well as long all-atom MD simulations (32), the latter showing that all four Trp41 residues flipped from the t-105 to the t90 rotamer after 500 ns. To further address this issue, we also calculated the PMF (Fig.…”

Application of solid-state NMR restraint potentials in membrane protein modeling

“…Identifying proper monomer structures greatly reduces the conformation space needed to be searched for corresponding oligomer structures. By clustering the determined oligomers based on total potential energies and crossing angles, we found that the M2TMP tetramer has strong preference of being left-handed, placing Ser31, His37, and Trp41 in the pore region, which is in good agreement with the previously determined structure [14,42] and the recent X-ray structure [6]. However, based on the χ 2 distributions of His37 and Trp41, four different conformations were identified.…”

“…These conformations yields different distances between His37 (i) and Trp41 (i+1) as well as between Trp41 (i) and Trp41 (i+1) , which dictates the relative orientations of both residues inside the pore; for example, based on PDB:1NYJ, these distances are (3.8 Å, 11.7 Å) in (t60,t90), (3.6 Å, 11.4 Å) in (t-160,t90), (3.8 Å, 5.1 Å) in (t-160,t-105), and (3.6 Å, 5.1 Å) in (t60,t-105), where the first one is for His37 (i) and Trp41 (i+1) and the second one for Trp41 (i) and Trp41 (i+1) . Three conformations corresponding to (t-160,t90), (t-160,t-105), and (t60,t90) have been determined by 19 F SSNMR [42], REDOR SSNMR [14] and MD simulations [43], respectively.…”

Structural basis for proton conduction and inhibition by the influenza M2 protein

“…All structures agreed on the relative positions of pore-facing versus lipid-facing side chains; however, key differences exist about the rotameric states of His37 and Trp41. 17,30,31,84 It is worth noting that side chain conformations cannot be obtained from orientedmembrane SSNMR experiments, as they only measure backbone NAH bond orientations. The His37 and Trp41 rotamers in the recent oriented-membrane SSNMR structure of M2(22-62) were based on computational modeling.…”