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Influenza changes channels Until recently, the pH-gated proton channel of influenza A virus, M2, was effectively targeted by amantadine-based antivirals, but resistance to these drugs is now widespread. Two groups now present structural studies of M2 proton channel. Jason Schnell and James Chou determine the structure of a 38-residue segment of M2, in complex with rimantadine, by NMR spectroscopy. Amanda Stouffer et al . determined the crystal structure of a 25-residue fragment of M2, with and without amantadine, using X-ray diffraction. Strikingly, the resulting structures suggest two very different mechanisms by which the drug inhibits the channel. The proposed mechanisms are discussed by Christopher Miller in an accompanying News & Views article. A vital component of influenza A virus' replication machinery is the M2 proton channel. Until recently, M2 was effectively targeted by amantadane-based antivirals, but resistance to these drugs is now so widespread that they have become ineffective. In the first of two related papers, the structure of a 38-residue segment of M2, in complex with rimantadine, is determined by NMR spectroscopy. It is concluded that a rimantadine molecule binds to each monomer at the protein–lipid interface and inhibits the tetrameric channel allosterically. The integral membrane protein M2 of influenza virus forms pH-gated proton channels in the viral lipid envelope 1 . The low pH of an endosome activates the M2 channel before haemagglutinin-mediated fusion. Conductance of protons acidifies the viral interior and thereby facilitates dissociation of the matrix protein from the viral nucleoproteins—a required process for unpacking of the viral genome 2 . In addition to its role in release of viral nucleoproteins, M2 in the trans-Golgi network (TGN) membrane prevents premature conformational rearrangement of newly synthesized haemagglutinin during transport to the cell surface by equilibrating the pH of the TGN with that of the host cell cytoplasm 3 . Inhibiting the proton conductance of M2 using the anti-viral drug amantadine or rimantadine inhibits viral replication 4 , 5 , 6 , 7 . Here we present the structure of the tetrameric M2 channel in complex with rimantadine, determined by NMR. In the closed state, four tightly packed transmembrane helices define a narrow channel, in which a ‘tryptophan gate’ is locked by intermolecular interactions with aspartic acid. A carboxy-terminal, amphipathic helix oriented nearly perpendicular to the transmembrane helix forms an inward-facing base. Lowering the pH destabilizes the transmembrane helical packing and unlocks the gate, admitting water to conduct protons, whereas the C-terminal base remains intact, preventing dissociation of the tetramer. Rimantadine binds at four equivalent sites near the gate on the lipid-facing side of the channel and stabilizes the closed conformation of the pore. Drug-resistance mutations are predicted to counter the effect of drug binding by either increasing the hydrophilicity of the pore or weakening helix–helix packing, thus facilitating channel opening.

The structure of the core region of the mitochondrial calcium uniporter (MCU) is determined by NMR and electron microscopy, revealing that MCU is a homo-pentamer with a specific transmembrane helix forming a hydrophilic pore across the membrane, and representing one of the largest membrane protein structures characterized by NMR spectroscopy. Mitochondrial calcium uniporter structure Many mitochondria use an inner membrane transporter, called the uniporter, to uptake and buffer large amounts of Ca 2+ . The MCU (mitochondrial calcium uniporter) is the pore-forming and Ca 2+ -conducting subunit of the uniporter, but its primary sequence does not resemble any calcium channel known to date. These authors report the structure of the core region of the MCU using nuclear magnetic resonance spectroscopy and electron microscopy. They show that the MCU is a homo-oligomer with a specific transmembrane helix forming a hydrophilic pore across the membrane. In addition to having a previously unknown channel architecture, the MCU is one of the largest structures to have been characterized by NMR spectroscopy. Mitochondria from many eukaryotic clades take up large amounts of calcium (Ca 2+ ) via an inner membrane transporter called the uniporter. Transport by the uniporter is membrane potential dependent and sensitive to ruthenium red or its derivative Ru360 (ref. 1 ). Electrophysiological studies have shown that the uniporter is an ion channel with remarkably high conductance and selectivity 2 . Ca 2+ entry into mitochondria is also known to activate the tricarboxylic acid cycle and seems to be crucial for matching the production of ATP in mitochondria with its cytosolic demand 3 . Mitochondrial calcium uniporter (MCU) is the pore-forming and Ca 2+ -conducting subunit of the uniporter holocomplex, but its primary sequence does not resemble any calcium channel studied to date. Here we report the structure of the pore domain of MCU from Caenorhabditis elegans , determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer in which the second transmembrane helix forms a hydrophilic pore across the membrane. The channel assembly represents a new solution of ion channel architecture, and is stabilized by a coiled-coil motif protruding into the mitochondrial matrix. The critical DXXE motif forms the pore entrance, which features two carboxylate rings; based on the ring dimensions and functional mutagenesis, these rings appear to form the selectivity filter. To our knowledge, this is one of the largest membrane protein structures characterized by NMR, and provides a structural blueprint for understanding the function of this channel.

Mitochondrial proton transport The transport of small molecules across the inner mitochondrial membrane is catalysed by a large family of membrane proteins called mitochondrial carriers. More than 40 different carriers have so far been identified to selectively translocate different substrates, but only one crystal structure is available — that of the bovine ADP/ATP carrier (ANT1). Now the structure of mitochondrial uncoupling protein 2 (UCP2), a member of the carrier family that translocates protons across the mitochondrial inner membrane, has been determined using a solution nuclear magnetic resonance (NMR) method. Its overall structure of resembles that of ANT1 — despite their low sequence identity — but the matrix side of the channel is substantially more open in UCP2. This method overcomes some of the challenges associated with using NMR spectroscopy to determine the structure of membrane proteins, so it seems likely that it will be possible to use the approach to solve the high-resolution NMR structures of other membrane proteins of comparable size. Mitochondrial uncoupling protein 2 (UCP2) is an integral membrane protein in the mitochondrial anion carrier protein family, the members of which facilitate the transport of small molecules across the mitochondrial inner membrane 1 , 2 . When the mitochondrial respiratory complex pumps protons from the mitochondrial matrix to the intermembrane space, it builds up an electrochemical potential 2 . A fraction of this electrochemical potential is dissipated as heat, in a process involving leakage of protons back to the matrix 2 . This leakage, or ‘uncoupling’ of the proton electrochemical potential, is mediated primarily by uncoupling proteins 2 . However, the mechanism of UCP-mediated proton translocation across the lipid bilayer is unknown. Here we describe a solution-NMR method for structural characterization of UCP2. The method, which overcomes some of the challenges associated with membrane-protein structure determination 3 , combines orientation restraints derived from NMR residual dipolar couplings (RDCs) and semiquantitative distance restraints from paramagnetic relaxation enhancement (PRE) measurements. The local and secondary structures of the protein were determined by piecing together molecular fragments from the Protein Data Bank that best fit experimental RDCs from samples weakly aligned in a DNA nanotube liquid crystal. The RDCs also determine the relative orientation of the secondary structural segments, and the PRE restraints provide their spatial arrangement in the tertiary fold. UCP2 closely resembles the bovine ADP/ATP carrier (the only carrier protein of known structure 4 ), but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Moreover, the nitroxide-labelled GDP binds inside the channel and seems to be closer to transmembrane helices 1–4. We believe that this biophysical approach can be applied to other membrane proteins and, in particular, to other mitochondrial carriers, not only for structure determination but also to characterize various conformational states of these proteins linked to substrate transport.

HIV-1 envelope spike (Env) is a type I membrane protein that mediates viral entry. We used nuclear magnetic resonance to determine an atomic structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in bicelles that mimic a lipid bilayer. The TM forms a well-ordered trimer that protects a conserved membrane-embedded arginine. An amino-terminal coiled-coil and a carboxyl-terminal hydrophilic core stabilize the trimer. Individual mutations of conserved residues did not disrupt the TM trimer and minimally affected membrane fusion and infectivity. Major changes in the hydrophilic core, however, altered the antibody sensitivity of Env. These results show how a TM domain anchors, stabilizes, and modulates a viral envelope spike and suggest that its influence on Env conformation is an important consideration for HIV-1 immunogen design.

The structure of the oligomeric hepatitis C virus viroporin p7 protein, solved by NMR spectroscopy, is reported; this protein can self-assemble into a channel complex that conducts cations and has a funnel-like channel architecture. Structure of a hepatitis virus cation channel Hepatitis C virus (HCV) is a major cause of liver diseases and cancer. With no protective vaccine available and therapeutic options still limited, it is important to explore new therapeutic targets. Here James Chou and colleagues report the structure of the oligomeric hepatitis C virus viroporin p7 protein, solved by NMR spectroscopy. This protein can self-assemble into a channel complex that conducts cations. A high-resolution view of the channel hexamer reveals novel funnel-like channel architecture and functional investigations identify residues important for channel activity. The hepatitis C virus (HCV) has developed a small membrane protein, p7, which remarkably can self-assemble into a large channel complex that selectively conducts cations 1 , 2 , 3 , 4 . We wanted to examine the structural solution that the viroporin adopts in order to achieve selective cation conduction, because p7 has no homology with any of the known prokaryotic or eukaryotic channel proteins. The activity of p7 can be inhibited by amantadine and rimantadine 2 , 5 , which are potent blockers of the influenza M2 channel 6 and licensed drugs against influenza infections 7 . The adamantane derivatives have been used in HCV clinical trials 8 , but large variation in drug efficacy among the various HCV genotypes has been difficult to explain without detailed molecular structures. Here we determine the structures of this HCV viroporin as well as its drug-binding site using the latest nuclear magnetic resonance (NMR) technologies. The structure exhibits an unusual mode of hexameric assembly, where the individual p7 monomers, i, not only interact with their immediate neighbours, but also reach farther to associate with the i +2 and i +3 monomers, forming a sophisticated, funnel-like architecture. The structure also points to a mechanism of cation selection: an asparagine/histidine ring that constricts the narrow end of the funnel serves as a broad cation selectivity filter, whereas an arginine/lysine ring that defines the wide end of the funnel may selectively allow cation diffusion into the channel. Our functional investigation using whole-cell channel recording shows that these residues are critical for channel activity. NMR measurements of the channel–drug complex revealed six equivalent hydrophobic pockets between the peripheral and pore-forming helices to which amantadine or rimantadine binds, and compound binding specifically to this position may allosterically inhibit cation conduction by preventing the channel from opening. Our data provide a molecular explanation for p7-mediated cation conductance and its inhibition by adamantane derivatives.

Membrane proteins are encoded by 20-35% of genes but represent <1% of known protein structures to date. Thus, improved methods for membrane-protein structure determination are of critical importance. Residual dipolar couplings (RDCs), commonly measured for biological macromolecules weakly aligned by liquid-crystalline media, are important global angular restraints for NMR structure determination. For α-helical membrane proteins >15 kDa in size, Nuclear-Overhauser effect-derived distance restraints are difficult to obtain, and RDCs could serve as the main reliable source of NMR structural information. In many of these cases, RDCs would enable full structure determination that otherwise would be impossible. However, none of the existing liquid-crystalline media used to align water-soluble proteins are compatible with the detergents required to solubilize membrane proteins. We report the design and construction of a detergent-resistant liquid crystal of 0.8-μm-long DNA-nanotubes that can be used to induce weak alignment of membrane proteins. The nanotubes are heterodimers of 0.4-μm-long six-helix bundles each self-assembled from a 7.3-kb scaffold strand and >170 short oligonucleotide staple strands. We show that the DNA-nanotube liquid crystal enables the accurate measurement of backbone NH and CαHα RDCs for the detergent-reconstituted ζ-ζ transmembrane domain of the T cell receptor. The measured RDCs validate the high-resolution structure of this transmembrane dimer. We anticipate that this medium will extend the advantages of weak alignment to NMR structure determination of a broad range of detergent-solubilized membrane proteins.

Acquired resistance to cetuximab in colorectal cancers is partially mediated by the acquisition of mutations located in the cetuximab epitope in the epidermal growth factor receptor (EGFR) ectodomain and hinders the clinical application of cetuximab. We develop a structure-guided and phage-assisted evolution approach for cetuximab evolution to reverse EGFR S492R - or EGFR G465R -driven resistance without altering the binding epitope or undermining antibody efficacy. Two evolved cetuximab variants, Ctx-VY and Ctx-Y104D, exhibit a restored binding ability with EGFR S492R , which harbors the most common resistance substitution, S492R. Ctx-W52D exhibits restored binding with EGFR harboring another common cetuximab resistance substitution, G465R (EGFR G465R ). All the evolved cetuximab variants effectively inhibit EGFR activation and downstream signaling and induce the internalization and degradation of EGFR S492R and EGFR G465R as well as EGFR WT . The evolved cetuximab variants (Ctx-VY, Ctx-Y104D and Ctx-W52D) with one or two amino acid substitutions in the complementarity-determining region inherit the optimized physical and chemical properties of cetuximab to a great extent, thus ensuring their druggability. Our data collectively show that structure-guided and phage-assisted evolution is an efficient and general approach for reversing receptor mutation-mediated resistance to therapeutic antibody drugs. Acquired resistance to cetuximab can be mediated by generation of mutations in the EGFR ectodomain. Here the authors report a structure-guided and phage-assisted evolution approach for cetuximab evolution to reverse resistance without altering the binding epitope or undermining antibody efficacy.

The membrane-proximal external region (MPER) of the HIV-1 envelope glycoprotein (Env) bears epitopes of broadly neutralizing antibodies (bnAbs) from infected individuals; it is thus a potential vaccine target. We report an NMR structure of the MPER and its adjacent transmembrane domain in bicelles that mimic a lipid-bilayer membrane. The MPER lies largely outside the lipid bilayer. It folds into a threefold cluster, stabilized mainly by conserved hydrophobic residues and potentially by interaction with phospholipid headgroups. Antigenic analysis and comparison with published images from electron cryotomography of HIV-1 Env on the virion surface suggest that the structure may represent a prefusion conformation of the MPER, distinct from the fusionintermediate state targeted by several well-studied bnAbs. Very slow bnAb binding indicates that infrequent fluctuations of the MPER structure give these antibodies occasional access to alternative conformations of MPER epitopes. Mutations in the MPER not only impede membrane fusion but also influence presentation of bnAb epitopes in other regions. These results suggest strategies for developing MPER-based vaccine candidates.

Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

Prediction of transmembrane helices (TMH) in alpha helical membrane proteins provides valuable information about the protein topology when the high resolution structures are not available. Many predictors have been developed based on either amino acid hydrophobicity scale or pure statistical approaches. While these predictors perform reasonably well in identifying the number of TMHs in a protein, they are generally inaccurate in predicting the ends of TMHs, or TMHs of unusual length. To improve the accuracy of TMH detection, we developed a machine-learning based predictor, MemBrain, which integrates a number of modern bioinformatics approaches including sequence representation by multiple sequence alignment matrix, the optimized evidence-theoretic K-nearest neighbor prediction algorithm, fusion of multiple prediction window sizes, and classification by dynamic threshold. MemBrain demonstrates an overall improvement of about 20% in prediction accuracy, particularly, in predicting the ends of TMHs and TMHs that are shorter than 15 residues. It also has the capability to detect N-terminal signal peptides. The MemBrain predictor is a useful sequence-based analysis tool for functional and structural characterization of helical membrane proteins; it is freely available at http://chou.med.harvard.edu/bioinf/MemBrain/.