The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidification of the interior of viral particles entrapped in endosomes. M2 is the target of the anti-influenza drugs amantadine and rimantadine; recently, resistance to these drugs in humans, birds and pigs has reached more than 90% (ref. 1). Here we describe the crystal structure of the transmembrane-spanning region of the homotetrameric protein in the presence and absence of the channel-blocking drug amantadine. pH-dependent structural changes occur near a set of conserved His and Trp residues that are involved in proton gating. The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses. Binding of amantadine physically occludes the pore, and might also perturb the pK(a) of the critical His residue. The structure provides a starting point for solving the problem of resistance to M2-channel blockers.

The changes that lead to activation of G protein-coupled receptors have not been elucidated at the structural level. In this work we report the crystal structures of both ground state and a photoactivated deprotonated intermediate of bovine rhodopsin at a resolution of 4.15 Å. In the photoactivated state, the Schiff base linking the chromophore and Lys-296 becomes deprotonated, reminiscent of the G protein-activating state, metarhodopsin II. The structures reveal that the changes that accompany photoactivation are smaller than previously predicted for the metarhodopsin II state and include changes on the cytoplasmic surface of rhodopsin that possibly enable the coupling to its cognate G protein, transducin. Furthermore, rhodopsin forms a potentially physiologically relevant dimer interface that involves helices I, II, and 8, and when taken with the prior work that implicates helices IV and V as the physiological dimer interface may account for one of the interfaces of the oligomeric structure of rhodopsin seen in the membrane by atomic force microscopy. The activation and oligomerization models likely extend to the majority of other G protein-coupled receptors.G protein-coupled receptor ͉ G protein-coupled receptor activation ͉ phototransduction ͉ membrane protein structure G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors in animals, accounting for Ϸ3% of the genome (1). GPCRs are involved in detecting a large variety of chemical and physical signals, and they are the targets of Ϸ50% of current therapeutics. Structural information on GPCRs has been limited because of difficulties with their expression, purification, intrinsic chemical heterogeneity, and instability. These biochemical problems were overcome by using rhodopsin as a model GPCR, as it is highly expressed in a homogeneous form in rod photoreceptors and stabilized in the ground state by its covalently bound chromophore, 11-cis-retinal (2).The first crystal structure of rhodopsin revealed the arrangement of helices, the interhelical connections, the chromophore binding site, the extracellular ''plug,'' interactions involved in ligand binding in other GPCRs, and cytoplasmic helix 8 (3). Further improvements in the rhodopsin crystals yielded higher-resolution diffraction data that provided details about the effects of water molecules located close to the chromophore and more precise descriptions of the cytoplasmic loops. However, the improved crystals did not elucidate the mechanism of activation (4, 5). The arrangement of the seven transmembrane helices of rhodopsin differs from that in the more completely structurally studied bacterial retinoid-binding protein, bacteriorhodopsin (6).Upon absorption of a single photon of light, rhodopsin's chromophore, 11-cis-retinal, isomerizes to form all-trans-retinal, a covalently bound, full agonist. Once all-trans-retinal is formed, the protein portion of rhodopsin progresses through a series of photostates, including bathorhodopsin, lumirhodopsin, and metarhodopsin I (Met...

The M2 proton channel from the influenza A virus is a small protein with a single transmembrane helix that associates to form a tetramer in vivo. This protein forms proton-selective ion channels, which are the target of the drug amantadine. Here, we propose a mechanism for the pH-dependent association, and amantadine binding of M2, based on studies of a peptide representing the M2 transmembrane segment in dodecylphosphocholine micelles. Using analytical ultracentrifugation, we find that the sedimentation curves for the peptide depend on its concentration in the micellar phase. The data are well-described by a monomer-tetramer equilibrium, and the binding of amantadine shifts the monomer-tetramer equilibrium toward tetrameric species. Both tetramerization and the binding of amantadine lead to increases in the magnitude of the ellipticity at 223 nm in the circular dichroism spectrum of the peptide. The tetramerization and binding of amantadine are more favorable at elevated pH, with a pK(a) that is assigned to a His side chain, the only ionizable residue within the transmembrane helix. Our results, interpreted quantitatively in terms of a reversible monomer and tetramer protonation equilibrium model, suggest that amantadine competes with protons for binding to the deprotonated tetramer, thereby stabilizing the tetramer in a slightly altered conformation. This model accounts for the observed inhibition of proton flux by amantadine. Additionally, our measurements suggest that the M2 tetramer is substantially protonated at neutral pH and that both singly and doubly protonated states could be involved in M2's proton conduction at more acidic pHs.

The M2 protein from influenza A virus is a 97-residue homotetrameric membrane protein that functions as a proton channel. To determine the features required for the assembly of this protein into its native tetrameric state, the protein was prepared by total synthesis using native chemical ligation of unprotected peptide segments. Circular dichroism spectroscopy of synthetic M2 protein in dodecylphosphocholine (DPC) micelles indicated that approximately 40 residues were in an alpha-helical secondary structure. The tetramerization of the full-length protein was compared to that of a 25-residue transmembrane (TM) fragment. Analytical ultracentrifugation demonstrated that both the peptide and the full-length protein in DPC micelles existed in a monomer-tetramer equilibrium. Comparison of the association constants for the two sequences showed the free energy of tetramerization of the full-length protein was more favorable by approximately 7 kcal/mol. Partial proteolysis of DPC-solubilized M2 was used as a further probe of the structure of the full-length protein. A 15-20-residue segment C-terminal to the membrane-spanning region was found to be highly resistant to digestion by chymotrypsin and trypsin. This region, which we have modeled as an extension of the TM helices, may help to stabilize the tetrameric assembly.