M2 is a 97-residue single-pass membrane protein with its N- and C-termini directed toward the outside and inside of the virion, respectively; it is a homotetramer in its native state8, 9
. The four TM helices form a channel in which His37 is the pH sensor and Trp41, the gate6, 10, 11
. The adamantane-based drugs, amantadine and rimantadine, which target the M2 channel, have been used as first-choice antiviral drugs against community outbreaks of influenza A viruses for many years, but resistance to the adamantanes has recently become widespread. Many structural models of this channel have been built, based on sequence analysis, mutagenesis, and solid-state NMR8, 11, 12
. Many of these studies have been done on inherently unstable TM-only constructs, however, leading to conflicting structural conclusions.
While the TM-only peptide fails to form a stable tetramer, a construct of residues 18–60 [M2(18–60)], that includes 15 residues of the C-terminus in addition to the TM region, forms a stable tetramer in dihexanoyl-phosphatidyl-choline (DHPC) detergent micelles and yields high-resolution NMR spectra (Supplementary Fig. S1
). In the closed conformation at pH 7.5, M2(18–60) is a homotetramer, in which each subunit has an unstructured N-terminus (residues 18–23), a channel-forming TM helix (residues 25–46), a short flexible loop (residues 47–50), and a C-terminal AP helix (residues 51–59). The TM helices assemble into a four-helix bundle with a left-handed twist angle of ~23° and a well-defined pore (). A ring of methyl groups from Val27 constricts the N-terminal end of the pore to ~3.1 Å (inner diameter). In agreement with proposed models11, 13
, His37 and Trp41 are inside the pore. A 3-bond, 15
Cγ scalar coupling (3JNCγ
) value of 1.5 Hz (Supplementary Table 1
), shows the His37 χ1
rotamer to be predominantly trans
, but with significant rotameric averaging. The χ1
of Trp41 is essentially locked in the trans
position, as determined by 3JNCγ
of 2.6 Hz, while the χ2
is also fixed at around −120° by the sidechain Hε1 – Nε1 dipolar coupling and NOEs. The Trp41 indole rings are at van der Waals (VDW) distance from each other, prohibiting passage of water or ions (). The indole Hε1 of one subunit is on average 3.5 Å from the Asp44 carboxyl carbon of the adjacent subunit. The two residues can form an intermolecular hydrogen bond that stabilizes the closed Trp41 gate. The sidechain of Arg45 likely participates in an intermolecular interaction with Asp44. These findings are consistent with the increased pH-modulated activity of channels in which asparagine has replaced Asp4414
. The C-terminal end of the channel extends into a loop (residues 47–50) that connects the TM domain to the C-terminal AP helix. RDCs and intra- and inter-monomer NOEs show that the AP helices lie roughly perpendicular (~82°) to the TM helices and assemble head-to-tail with a right-handed packing mode to form the base of the channel. The orientation and amphipathic character of the AP helices suggest that the C-terminal base lies on the surface of the membrane.
Figure 1 Structure of the M2 channel. a, An ensemble of 15 low energy structures derived from NMR restraints. Since residues 47–50 are unstructured, the TM helices (residues 25–46) and the AP helices (residues 51–59) are superimposed separately. (more ...)
Residues 47–50 give no NOE peaks and have no stable, hydrogen-bonded structure in the detergent micelles used in our work. We believe that this segment adopts a more stable conformation in the viral membrane because Cys50, which we mutated to serine to avoid disulfide formation, is normally palmitoylated15
. Modeling shows that extending the TM helix to Phe48 would place residue 50 facing the membrane, allowing for insertion of the palmitoyl acyl chain into the lipid bilayer. This minor rearrangement would also move the AP helices closer to the TM domain.
Drug binding has the effect of stabilizing the closed conformation. Upon addition of drug, the resonances of residues 43–46 at the C-terminus of the channel, which are severely exchange-broadened in the drug-free sample, became significantly sharper and more homogeneous (Supplementary Fig. S2
). The protein-drug NOEs collected from four different NOESY spectra (Supplementary Fig. S3
) place the binding site between adjacent helices at the C-terminal end of the TM domain near the Trp41 gate, on the membrane side of the channel (). We could not detect drug NOEs in other parts of the protein, including the widely proposed drug-binding site in the pore of the channel. The amine head-group of rimantadine is in contact with the polar sidechains of Asp44, Arg45, and the indole amine of Trp41. The sidechains of Ile42 from one helix, and Leu40 and Leu43 from another helix form the hydrophobic walls of the binding pocket that interact with the adamantane group of rimantadine. Thus, rimantadine covers a unique polar patch in the otherwise hydrophobic environment of the TM domain. Interactions between rimantadine and the channel are consistent with structure-activity relationships of the adamantane group16
. In particular, the basic nitrogen group and size limits at the methyl site are critical. These requirements are the result of the interactions with Asp44 and the small hydrophobic pocket around Ile42, respectively.
Water NOEs measured in the 110 ms 15N-separated NOESY experiments give a clear picture of water distribution relative to the channel (). The lipid-facing surface of the TM region is largely protected from water by the DHPC micelle. In the closed channel pore, the Val27 ring at the N-terminus and the Trp41 gate at the C-terminus essentially block water from freely diffusing into the pore from either side of the membrane. Within the TM region, only the amides of Ser31 and Ile32 have NOE crosspeaks at the chemical shift of water, probably corresponding to the hydroxyl proton of Ser31 in exchange with water. A polar residue is present at position 31 in all sequenced variants of M2, suggesting that proton conduction requires water bound to this site. This water may serve to bridge the proton relay from the N-terminal end of the pore to the His37 pH sensor. Water was detected at the C-terminus of the TM region, beginning at Arg45. The Hε1 of the Trp41 indole ring, which points toward the C-terminal side of the pore, also has a strong NOE to water, indicating that the base of the channel is accessible to bulk water.
Figure 2 Water accessibility of the M2 channel. a, Distribution of water NOEs relative to the structure. Amide protons colored in blue have a NOE crosspeak to water. Those that do not are colored red. b, The pore surface calculated using the program HOLE. The (more ...)
Lowering the pH from 7.5 to 6.5 broadens most of the NMR resonances corresponding to the TM helix (). The resonance broadening could not be attributed to protein aggregation, as the self-diffusion coefficients were essentially unchanged between pH 7.5 and 6.5. Thus, activation of the channel is coupled to increased conformational exchange in the TM domain. In contrast, the resonances of the AP helices are essentially unaffected by lowering the pH, indicating that the C-terminal base of the tetramer remains intact as the channel opens.
Figure 3 Low-pH induced destabilization of the channel and opening of the Trp41 gate. a, 1H-15N TROSY spectra of reconstituted M2(18–60) tetramer at pH 6.0, 6.5, 7.0, and 7.5, in the absence of rimantadine, recorded at 500 MHz 1H frequency and 30 °C. (more ...)
In addition to destabilizing helix-helix packing in the TM domain, channel activation must also correlate with increased dynamics of the Trp41 gate. Since the indole amide resonance of Trp41 remained intense as the pH was lowered from 7.5 to 6.0, it serves as a useful NMR probe for monitoring channel opening. We compared the ms timescale dynamics of the Trp41 indole ring between the closed and open states by carrying out relaxation-compensated Carr-Purcell-Meiboom-Gill (CPMG) experiments17
at pH 7.5, 7.0, and 6.0. A two-site exchange model fits the dependence of 15
N relaxation due to chemical shift exchange on the frequency of refocusing (1/τcp
) of chemical shift evolution (), implying that the gate switches between two configurations at any given pH. As the pH was lowered from 7.5 to 6.0, the rate of fluctuation increased by more than four-fold (), indicating that channel activation “unlocks” the gate. Adding rimantadine to the channel at an intermediate pH of 7.0 slowed the timescale of the gate motion to nearly that of a drug-free gate at pH 7.5 (). These results confirm that the reconstituted channels in the NMR sample are pH-gated, and are consistent with the location of the rimantadine site proximal to the gate.
The structure of the M2 proton channel thus reveals a simple yet effective gating. The tight packing of the four TM helices brings the bulky indole rings of Trp41 into VDW contact to form the channel gate. The gate is further stabilized by inter-subunit hydrogen bonds with Asp44. Lowering the pH protonates the imidazole rings of His37, destabilizing helix-helix packing by electrostatic repulsion. This conformational rearrangement breaks interactions between Trp41 and Asp44 and allows the gate to flip open. A pair of conserved N-terminal cysteines have been shown to form intermolecular disulfides in vivo9
. Thus, the TM helices are tethered at one end by N-terminal disulfides and at the other end by the C-terminal base, ensuring that destabilization of the four-helix bundle during channel activation does not cause dissociation of the tetramer (). Indeed, truncation of the AP helix results in channels that rapidly lose channel activity18
Figure 4 Schematic illustration of M2 channel activation. At high pH, the TM helices are packed tightly and the tryptophan gate is locked through intermolecular interactions with Asp44. At low pH, protonation of the His37 imidazoles destabilizes the TM helix packing, (more ...)
The external drug-binding site was unexpected. Drug-resistance mutations seemed to suggest a site inside the pore, because in early models of the channel, residues that lead to drug resistance were predicted to be pore-lining. The known mutations that confer drug resistance are L26F, V27A, A30T, S31N, G34E, and L38F. Mapping these residues onto the structure (Supplementary Fig. S7
) reveals that Val27, Ala30, and Gly34 are pore-lining, but Leu26, Ser31 and Leu38 are in the helix-helix packing interface. Moreover, these mutations are spread out over more than three turns of the TM helix, covering a distance much larger than the dimensions of amantadine or rimantadine. Pinto and Lamb have pointed out that having a cork-plugging-the-bottle model is insufficient to explain all the results of electrophysiology studies19
. For example, drug inhibition is more effective when applied to the closed channel than the open channel, which is not expected of a pore-blocking mechanism5
. Several of the drug-resistance mutations in pore-lining residues have been shown to retain drug binding20
. Although a pore blocker is expected to fit tightly in the pore, channel inhibition is unusually tolerant of modifications to the adamantane scaffold16
. Together, the above observations suggest an allosteric inhibition mechanism.
Is the external binding site consistent with all drug-resistance mutations? Although the exact structural effects of resistance mutations are difficult to predict, what they do have in common is that they either perturb the helix-helix interface (L26F, V27A, S31N, L38F), or increase the hydrophilicity of the pore (A30T, G34E). From this observation, and from detection of a conformational exchange among multiple states at lowered pH, we propose an allosteric inhibition mechanism that can account for all of the mutations. In our model, drug binding makes the closed channel harder to open, while drug-resistance mutations destabilize the closed channel, making it easier to open. Replacing Val27 with alanine enlarges the N-terminal opening and weakens helix-helix packing, and therefore may facilitate channel opening. Ala30 and Gly34 are inside the pore, and replacing them with threonine and glutamate, respectively, may facilitate pore hydration, and in turn, channel opening. Leu26, Ser31 and Leu38 are helix-helix interface residues; their mutations likely perturb helix-helix packing and lower the energetic cost of channel opening.
Why then, are no drug-resistant mutations observed near the drug-binding site? In fact, few mutations are ever observed in this region of the channel (drug-resistant or otherwise), which is not surprising due to the functional constraints placed on these residues in proximity to the channel pH sensor (His37) and the channel gate (Trp41). As the structure illustrates, intermolecular contacts between Asp44 and Arg45 form an integral part of the channel gate, along with Trp41. The residues that form the hydrophobic walls of the binding pocket – Leu40, Ile42, and Leu43 – are on the lipid face of the channel and must retain hydrophobicity for membrane partitioning. In order to accommodate the Trp41 indole rings within the channel, the helices splay slightly at the C-terminus of the TM domain, and interhelical contacts below Leu38 – with the exception of the Trp41 sidechains – are no longer important for channel assembly. Thus, residues essential to channel assembly are in the N-terminal half of the TM helix, exactly where drug-resistance mutations occur.
Binding from the membrane side is consistent with the high membrane partition coefficient of adamantane drugs, which effectively concentrates them in the membrane and lowers their level in the aqueous phase21, 22
. Adamantanes interact with a number of other ion channels, including viroporins from hepatitis C23
, the potassium channel Kcv of the chlorella virus PBCV-124
, and the human NMDA receptors25
. Hanatoxin, an allosteric inhibitor of voltage gated K+
channels with a high membrane partition coefficient, also has an external binding site26, 27
. Membrane-side binding may thus be a feature of many channel inhibitors. This mode of inhibition could be advantageous for drug design because drug molecules are typically much larger than hydrated ions selected by ion channels, and therefore the energy barrier for drug to find a blocking site inside the channel pores would be much higher than targeting a functional site from the membrane side of the channel.