Apoptosis requires recruitment of caspases by receptor-associated adaptors through homophilic interactions between the CARDs (caspase recruitment domains) of adaptor proteins and prodomains of caspases. We have solved the CARD structure of the RAIDD adaptor protein that recruits ICH-1/caspase-2. It consists of six tightly packed helices arranged in a topology homologous to the Fas death domain. The surface contains a basic and an acidic patch on opposite sides. This polarity is conserved in the ICH-1 CARD as indicated by homology modeling. Mutagenesis data suggest that these patches mediate CARD/CARD interaction between RAIDD and ICH-1. Subsequent modeling of the CARDs of Apaf-1 and caspase-9, as well as Ced-4 and Ced-3, showed that the basic/acidic surface polarity is highly conserved, suggesting a general mode for CARD/CARD interaction.

Mitochondria from multiple, eukaryotic clades uptake and buffer large amounts of calcium (Ca2+) via an inner membrane transporter called the uniporter. Early studies demonstrated that this transport requires a mitochondrial membrane potential and that the uniporter is itself Ca2+ activated, and blocked by ruthenium red or Ru3601. Later, electrophysiological studies demonstrated that the uniporter is an ion channel with remarkably high conductance and selectivity2. Ca2+ entry into mitochondria is also known to activate the TCA cycle and appears to be critical for matching ATP production in mitochondria with its cytosolic demand3. MCU (mitochondrial calcium uniporter) is the pore forming and Ca2+ conducting subunit of the uniporter, but its primary sequence does not resemble any calcium channel known to date. Here, we report the structure of the core region of MCU, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer with the second transmembrane helix forming 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 in the mitochondrial matrix. The critical DxxE motif forms the pore entrance featuring two carboxylate rings, which appear to be the selectivity filter based on the ring dimensions and functional mutagenesis. To our knowledge, this is one of the largest structures characterized by NMR, which provides a structural blueprint for understanding the function of this channel.

We report a magic angle spinning (MAS) NMR structure of the drug-resistant S31N mutation of M218–60 from Influenza A. The protein was dispersed in diphytanoyl-sn-glycero-3-phosphocholine lipid bilayers, and the spectra and an extensive set of constraints indicate that M218–60 consists of a dimer of dimers. In particular, ~280 structural constraints were obtained using dipole recoupling experiments that yielded well-resolved 13C–15N, 13C–13C, and 1H–15N 2D, 3D, and 4D MAS spectra, all of which show cross-peak doubling. Interhelical distances were measured using mixed 15N/13C labeling and with deuterated protein, MAS at ωr/2π = 60 kHz, ω0H/2π = 1000 MHz, and 1H detection of methyl–methyl contacts. The experiments reveal a compact structure consisting of a tetramer composed of four transmembrane helices, in which two opposing helices are displaced and rotated in the direction of the membrane normal relative to a four-fold symmetric arrangement, yielding a two-fold symmetric structure. Side chain conformations of the important gating and pH-sensing residues W41 and H37 are found to differ markedly from four-fold symmetry. The rmsd of the structure is 0.7 Å for backbone heavy atoms and 1.1 Å for all heavy atoms. This two-fold symmetric structure is different from all of the previous structures of M2, many of which were determined in detergent and/or with shorter constructs that are not fully active. The structure has implications for the mechanism of H+ transport since the distance between His and Trp residues on different helices is found to be short. The structure also exhibits two-fold symmetry in the vicinity of the binding site of adamantyl inhibitors, and steric constraints may explain the mechanism of the drug-resistant S31N mutation.

The tetrameric M2 proton channel from influenza A virus conducts protons at low pH and is inhibited by aminoadamantyl drugs such as amantadine and rimantadine. We report magic angle spinning NMR spectra of POPC and DPhPC membrane embedded M2 , both apo and in the presence of rimantadine. Similar linewidths in spectra of apo and bound M2 indicate that rimantadine does not have a significant impact on the dynamics or conformational heterogeneity of this construct. Substantial chemical shift changes for many residues in the transmembrane region support an allosteric mechanism of inhibition. A Rmt titration supports a binding stoichiometry of >1 Rmt molecule per channel while showing that nonspecific bindin0g or changes in membrane composition are unlikely sources of the chemical shift changes. In addition, doubling of spectral lines in all of the observed samples provides evidence that the channel assembles with twofold symmetry. The M2 protein from influenza A virus is a single pass membrane protein that assembles as a tetramer to form a H + selective channel that functions at low pH and is critical in the viral lifecycle. A class of aminoadamantyl inhibitors has become ineffective against many influenza strains due to mutations in the N-terminal region of the channel 1 , thus stimulating great interest in identification of the pharmacologically relevant binding site and the mechanism of inhibition and drug resistance. Discussion of an external, lipid-facing site and a pore-blocking site is ongoing 2-8 . A solution NMR structure 7 in DHPC micelles of M2 showed an external binding site at D44 via direct NOE measurement; however, the pharmacological relevance of this binding pocket was questioned due to possible detergent rgg@mit.edu. Supporting Information Available: Sample preparation and synthesis, description of assignments, additional spectra. This material is available free of charge via the Internet at http://pubs.acs.org has indistinguishable conduction compared to the full length protein 5 . It is presently unclear whether the discrepancy between the two observed binding sites arises due to detergent effects, the highly truncated construct, or other factors. We therefore initiated investigations of the fully functional construct, M2 18-60 in lipid bilayers with magic angle spinning (MAS) NMR. Our chemical shift data reveal global conformational changes upon drug binding that suggest an allosteric mechanism of inhibition, and peak doubling, indicating a twofold symmetric tetramer. NIH Public AccessFigure 1 (top) shows a 15 N-13 C one-bond zf-TEDOR 11,12 correlation spectrum of M2 18-60 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, demonstrating spectral assignments in both the absence (red) and presence (blue) of the inhibitor rimantadine (Rmt). Linewidths of ~1 ppm for both 15 N and 13 C are observed at 700 MHz for both drug-bound and apo samples, indicating conformational homogeneity. This narrow linewidth also indicates that the dynamics of this system are favorable for ...

Highlights d Transmembrane helix of death receptor 5 oligomerizes to drive downstream signaling d The transmembrane helix in lipid bilayer forms dimer-trimer interaction network d Receptor ectodomain in pre-ligand state inhibits receptor clustering and activation d Ligand binding overcomes the pre-ligand autoinhibition

SUMMARY Fas (CD95, Apo-1, TNFRSF6) is a prototypical apoptosis-inducing death receptor in the tumor necrosis factor receptor (TNFR) superfamily. While the extracellular domains of TNFRs form trimeric complexes with their ligands and the intracellular domains engage in higher-order oligomerization, the role of the transmembrane (TM) domains is unknown. Here, we determined the nuclear magnetic resonance (NMR) structures of mouse and human Fas-TM domains in bicelles that mimic lipid bilayers. Surprisingly, these domains use proline motifs to create optimal packing in homotrimer assembly distinct from classical trimeric coiled-coils in solution. Cancer-associated and structure-based mutations in Fas-TM disrupt trimerization in vitro and reduce apoptosis induction in vivo, indicating the essential role of intramembrane trimerization in receptor activity. Our data suggest that the structures represent the signaling-active conformation of Fas-TM, which appears to be different from the pre-ligand conformation. Analysis of other TNFR sequences suggests proline-containing sequences as common motifs for receptor TM trimerization.

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 fusion-intermediate 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.

At the United Nations Framework Convention on Climate Change Conference in Cancun, in November 2010, the Heads of State reached an agreement on the aim of limiting the global temperature rise to 2°C relative to preindustrial levels. They recognized that long-term future warming is primarily constrained by cumulative anthropogenic greenhouse gas emissions, that deep cuts in global emissions are required, and that action based on equity must be taken to meet this objective. However, negotiations on emission reduction among countries are increasingly fraught with difficulty, partly because of arguments about the responsibility for the ongoing temperature rise. Simulations with two earth-system models (NCAR/CESM and BNU-ESM) demonstrate that developed countries had contributed about 60-80%, developing countries about 20-40%, to the global temperature rise, upper ocean warming, and sea-ice reduction by 2005. Enacting pledges made at Cancun with continuation to 2100 leads to a reduction in global temperature rise relative to business as usual with a 1/3-2/3 (CESM 33-67%, BNU-ESM 35-65%) contribution from developed and developing countries, respectively. To prevent a temperature rise by 2°C or more in 2100, it is necessary to fill the gap with more ambitious mitigation efforts.climate modeling | Coupled Model Intercomparison Project phase 5 | Cancun pledge | climate ethics | geoengineering T he impact of human activities on climate change at global and regional scales, including surface temperature (1), sea-level pressure (2), tropopause height (3), precipitation (4), and ocean heat content (5), has been explored and assessed. Greenhouse gas emissions, mostly CO 2 , are the most important anthropogenic forcing on climate (6). The contribution of greenhouse gas emissions varies widely among nations in both the past and the future. As a result, the United Nations Framework Convention on Climate Change (UNFCCC) reached an agreement that each nation should accept its "common but differentiated responsibilities." This ethical construct demands attribution studies of the historical contribution of emissions to climate change (7). To date, research has tracked the causal chain of climate change from human activities to greenhouse gas emissions, to radiative forcing, and finally to climate change. However, this conventional methodological flow does not consider the reverse process or include feedbacks from climate change to greenhouse-gas concentrations via biogeochemistry or decision-making processes (8). More than 100 countries have adopted a global warming limit of 2°C or below (relative to preindustrial levels) as a guiding principle for mitigation efforts to reduce climate-change risks, impacts, and damage (9, 10). The relationship between the climate policy making and the 2°C target by an appropriate emission pathway has been studied in simple climate models and probabilistic analysis (11, 12). However, climate projection experiments under many emission scenarios, even the latest representative concentration pathways (RC...