The Influenza M2 Ectodomain Regulates the Conformational Equilibria of the Transmembrane Proton Channel: Insights from Solid-State Nuclear Magnetic Resonance

Abstract

The influenza M2 protein is the target of the amantadine family of antiviral drugs, and its transmembrane (TM) domain structure and dynamics have been extensively studied. However, little is known about the structure of the highly conserved N-terminal ectodomain, which contains epitopes targeted by influenza vaccines. In this study, we synthesized an M2 construct containing the N-terminal ectodomain and the TM domain, to understand the site-specific conformation and dynamics of the ectodomain and to investigate the ectodomain effect on the TM structure. We incorporated 13C, 15N-labeled residues into both domains and measured their chemical shifts and linewidths using solid-state NMR. The data indicate that the entire ectodomain is unstructured and dynamic, but the motion is slower for residues closer to the TM domain. 13C lineshapes indicate that this ecto-TM construct undergoes fast uniaxial rotational diffusion, similar to the isolated TM peptide, but drug binding increases the motional rates of the TM helix while slowing down the local motion of the ectodomain residues that are close to the TM domain. Moreover, 13C and 15N chemical shifts indicate that the ectodomain shifts the conformational equilibria of the TM residues towards the drug-bound state even in the absence of amantadine, thus providing a molecular structural basis for the lower inhibitory concentration of full-length M2 compared to the ectodomain-truncated M2. We propose that this conformational selection may result from electrostatic repulsion between negatively charged ectodomain residues in the tetrameric protein. Together with the recent study of the M2 cytoplasmic domain, these results show that intrinsically disordered extramembrane domains in membrane proteins can regulate functionally relevant conformation and dynamics of the structurally ordered transmembrane domains.

Influenza A Virus M2 Protein: Roles from Ingress to Egress

“…These studies suggest that the ED conformation depends on the membrane composition. In DMPC bilayers, some residues show a β-strand conformation that shifts to an α-helical conformation in the presence of cholesterol (mimicking the viral membrane) [ 74 , 75 ]. It is worthwhile to mention that crystallography of M2 ED—Fab complex also suggested that M2-ED exists in different conformations supporting the notion that M2-ED is intrinsically disordered [ 76 , 77 ].…”

“…The post-APH CT up to residue 71 is largely unstructured with random coil conformation, highly mobile and, unlike the ED, insensitive to the membrane composition [ 75 , 78 ]. Together, these studies suggest that the presence of either the ED or CT favors TMD conformation towards a more stable, resembling drug bound state [ 74 , 75 , 78 ]. Moreover, the post-APH CT also changes the TMD conformation to one favoring proton binding, though its deletion does not affect the ion channel activity [ 74 ].…”

“…Together, these studies suggest that the presence of either the ED or CT favors TMD conformation towards a more stable, resembling drug bound state [ 74 , 75 , 78 ]. Moreover, the post-APH CT also changes the TMD conformation to one favoring proton binding, though its deletion does not affect the ion channel activity [ 74 ].…”

Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy

“…These studies showed that the conduction-relevant structure of M2 depends markedly on the membrane environment [18], but the conduction properties are insensitive to the construct length beyond the TM domain if the same membrane environment is used [19]. Several M2 constructs have been used in biophysical studies because of the modular sequence and multifunctional nature of this protein: a highly conserved and disordered N-terminal ectodomain [20] mediates protein incorporation into the virion [21] and is the target of universal influenza vaccines [22]. The TM domain (residues 22-46) is necessary and sufficient to exhibit proton conductance that is similar to full-length M2 [19].…”

The L46P Mutant Confers a Novel Allosteric Mechanism of Resistance Toward the Influenza A Virus M2 S31N Proton Channel Blockers

“…The AM2 channel is a homotetrameric transmembrane protein with 97 residues per monomer. The N-terminal domain (residues 1-23) is largely unstructured with polar residues that help increase the hydration of the pore to facilitate proton conductance (Kwon and Hong, 2016;Ma and Wang, 2018) and for incorporation into virions (Park et al, 1998). The transmembrane (TM) domain (residues 24-43) is required for the formation of a left-handed 4-helix bundle (Cady and Hong, 2008;Stouffer et al, 2008) and for both proton conductance and selectivity (Balannik et al, 2010) as well as drug binding (Ma et al, 2009).…”