Mechanisms of proton conduction and gating in influenza M2 proton channels from solid-state NMR

Abstract

The M2 protein of influenza viruses forms an acid-activated tetrameric proton channel. We used solid-state nuclear magnetic resonance spectroscopy to determine the structure and functional dynamics of the pH-sensing and proton-selective histidine-37 in M2 bound to a cholesterol-containing virus-envelope-mimetic membrane so as to better understand the proton conduction mechanism. In the high-pH closed state, the four histidines form an edge-face π-stacked structure, preventing the formation of a hydrogen-bonded water chain to conduct protons. In the low-pH conducting state, the imidazoliums hydrogen-bond extensively with water and undergo microsecond ring reorientations with an energy barrier greater than 59 kilojoules per mole. This barrier is consistent with the temperature dependence of proton conductivity, suggesting that histidine-37 dynamically shuttles protons into the virion. We propose a proton conduction mechanism in which ring-flip-assisted imidazole deprotonation is the rate-limiting step.

Fig. 1

¹⁵N and ¹³C chemical shifts of His³⁷-labeled M2TM in viral membranes reveal pH-dependent imidazole protonation state and tautomeric structures. (A and B) 2D ¹³C-¹³C correlation spectra, (A) pH 8.5 and (B) pH 4.5. The τ- and π-tautomer peaks are assigned in red and blue, and the charged His³⁷ peaks are in green. (C and D) 2D ¹⁵N-¹³C correlation spectra, (C) pH 8.5 and (D) pH 4.5. (E) Summary of the imidazole chemical shifts.

Fig. 2

His³⁷ rotameric conformation from Cα-Nδ1 distances. (A) pH 8.5 data, with representative rotational-echo double-resonance control (S₀), dephased (S), and difference (∆S) spectra. The 3.9 Å distance indicates χ₂ = 180°. (B) pH 4.5 data, showing a similar distance and χ₂ angle. (C) Top and side views of the His³⁷ tetrad in the tt rotamer in the high-pH structure [Protein Data Bank (PDB) number 2KQT] (22). (D) Top view of the His³⁷ tetrad in the tt rotamer in the low-pH structure (PDB number 3C9J) (13).

Fig. 3

His³⁷ sidechains reorient at low pH but remain static at high pH at physiological temperature. (A) 303 K ¹³C-¹⁵N dipolar couplings. At pH 8.5, a 1:1 combination of Cγ-Nδ1 and Cε1-Nδ1 couplings reaches the rigid limit. At pH 4.5, the dominant Cγ-Nδ1 coupling is motionally averaged. (B) Cδ2-Hδ2 coupling at 308 K is motionally averaged at pH 4.5 but in the rigid limit at pH 8.5. (C) Measured order parameters at pH 4.5. (D) Two-site jump of imidazolium at low pH. A 45° reorientation around the Cβ-Cγ bond fits the observed order parameters.

Fig. 4

Charged His³⁷ H-bonds with water and undergoes ring flips to relay protons. (A) N-H dipolar couplings at 243 K. At pH 8.5, Nε2(τ) is not H-bonded, whereas Nδ1(π) is. The unprotonated Nδ1(τ) shows a weak H-bond. At pH 4.5, both nitrogens show weak couplings and bond stretching. (B) C-H dipolar couplings of Cδ2 and Cε1 at 243 K. The Cε1-Hε1 bond is stretched, whereas Cδ2-Hδ2 is not. (C) Imidazole bond lengths and H-bond networks at high and low pH. (D) His³⁷ structure and dynamics and proposed water orientations across the tetrad. Trp41 may interact with His³⁷ at high pH_out. (E) Proposed imidazole structural changes in a cycle in which multiple ring reorientations mediate the transfer of two protons.