Serotonin receptors (5-HT3AR) directly regulate gut movement, and drugs that inhibit 5-HT3AR function are used to control emetic reflexes associated with gastrointestinal pathologies and cancer therapies. The 5-HT3AR function involves a finely tuned orchestration of three domain movements that include the ligand-binding domain, the pore domain, and the intracellular domain. Here, we present the structure from the full-length 5-HT3AR channel in the apo-state determined by single-particle cryo-electron microscopy at a nominal resolution of 4.3 Å. In this conformation, the ligand-binding domain adopts a conformation reminiscent of the unliganded state with the pore domain captured in a closed conformation. In comparison to the 5-HT3AR crystal structure, the full-length channel in the apo-conformation adopts a more expanded conformation of all the three domains with a characteristic twist that is implicated in gating.

The transient receptor potential vanilloid 5 (TRPV5) channel is a member of the transient receptor potential (TRP) channel family, which is highly selective for Ca2+, that is present primarily at the apical membrane of distal tubule epithelial cells in the kidney and plays a key role in Ca2+ reabsorption. Here we present the structure of the full-length rabbit TRPV5 channel as determined using cryo-EM in complex with its inhibitor econazole. This structure reveals that econazole resides in a hydrophobic pocket analogous to that occupied by phosphatidylinositides and vanilloids in TRPV1, thus suggesting conserved mechanisms for ligand recognition and lipid binding among TRPV channels. The econazole-bound TRPV5 structure adopts a closed conformation with a distinct lower gate that occludes Ca2+ permeation through the channel. Structural comparisons between TRPV5 and other TRPV channels, complemented with molecular dynamics (MD) simulations of the econazole-bound TRPV5 structure, allowed us to gain mechanistic insight into TRPV5 channel inhibition by small molecules.

Serotonin receptor (5-HT 3A R) 1 , a cationic pentameric ligand-gated ion channel (pLGIC), is the clinical target for management of nausea and vomiting associated with radiation and chemotherapies 2 . Upon binding, serotonin induces a global conformational change encompassing the ligand-binding extracellular domain (ECD), the transmembrane domain (TMD), and the intracellular domain (ICD), the molecular details of which are unclear. Here, we present two serotonin-bound structures of the full-length 5-HT 3A R in distinct conformations at 3.32 Å and 3.89 Å resolutions that reveal the mechanism underlying channel activation. When compared to Apo-5-HT 3A R, serotonin-bound states underwent a large twisting motion in the ECD and TMD leading to the opening of a 165 Å long permeation pathway. Notably, this motion results in creation of lateral portals for ion permeation at the interface of the TMD and ICD. Combined with molecular dynamics simulations, these structures provide novel insights into conformational coupling across domains and functional modulation.

Desensitization in pentameric ligand-gated ion channels plays an important role in regulating neuronal excitability. Here, we show that docosahexaenoic acid (DHA), a key ω−3 polyunsaturated fatty acid in synaptic membranes, enhances the agonist-induced transition to the desensitized state in the prokaryotic channel GLIC. We determined a 3.25 Å crystal structure of the GLIC-DHA complex in a potentially desensitized conformation. The DHA molecule is bound at the channel-periphery near the M4 helix and exerts a long-range allosteric effect on the pore across domain-interfaces. In this previously unobserved conformation, the extracellular-half of the pore-lining M2 is splayed open, reminiscent of the open conformation, while the intracellular-half is constricted, leading to a loss of both water and permeant ions. These findings, in combination with spin-labeling/EPR spectroscopic measurements in reconstituted-membranes, provide novel mechanistic details of desensitization in pentameric channels.DOI: http://dx.doi.org/10.7554/eLife.23886.001

dThe subunit of bacterial F 1 F O ATP synthases plays an important regulatory role in coupling and catalysis via conformational transitions of its C-terminal domain. Here we present the first low-resolution solution structure of of Mycobacterium tuberculosis (Mt) F 1 F O ATP synthase and the nuclear magnetic resonance (NMR) structure of its C-terminal segment (Mt 103-120 ). Mt is significantly shorter (61.6 Å) than forms of the subunit in other bacteria, reflecting a shorter C-terminal sequence, proposed to be important in coupling processes via the catalytic ␤ subunit. The C-terminal segment displays an ␣-helical structure and a highly positive surface charge due to the presence of arginine residues. Using NMR spectroscopy, fluorescence spectroscopy, and mutagenesis, we demonstrate that the new tuberculosis (TB) drug candidate TMC207, proposed to bind to the proton translocating c-ring, also binds to Mt. A model for the interaction of TMC207 with both and the c-ring is presented, suggesting that TMC207 forms a wedge between the two rotating subunits by interacting with the residues W15 and F50 of and the c-ring, respectively. T19 and R37 of provide the necessary polar interactions with the drug molecule. This new model of the mechanism of TMC207 provides the basis for the design of new drugs targeting the F 1 F O ATP synthase in M. tuberculosis.

Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyRs) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structures have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to channel function are still unanswered. Here, we present Cryo-EM structures of the full-length GlyR protein complex reconstituted into lipid nanodiscs that are captured in the unliganded (closed), glycine-bound (open and desensitized), and allosteric modulator-bound conformations. A comparison of these states reveals global conformational changes underlying GlyR channel gating and modulation. The functional state assignments were validated by molecular dynamics simulations, and the observed permeation events are in agreement with the anion selectivity and conductance of GlyR. These studies provide the structural basis for gating, ion selectivity, and single-channel conductance properties of GlyR in a lipid environment.

validate structural predictions using solution-phase small angle neutron scattering (SANS). We have acquired SANS data from GLIC under nonactivating buffer conditions, hiding the detergent micelle by use of deuteration. Using structures and molecular dynamics simulations of different states, the agreement with the SANS data has been evaluated. Although differences in the fit of the predicted models to the experimental data were small, our work shows that a resting state model is consistent with the room-temperature experimental data. This work shows the potential for using SANS in conjunction with other methods in the study of membrane proteins, while underlining the importance of high quality SANS data of multiple states. . Serotonin receptors (5-HT 3A R) are cation-selective, neurotransmitter-gated, pentameric ion channels that play a crucial role in regulating cellular excitability. 5-HT 3A R activities directly regulate gut movement and therefore drugs that inhibit 5-HT 3A R function are used to control vomiting reflexes. Serotonin binding induces a global conformational change encompassing the ligandbinding extracellular domain (ECD), the transmembrane domain (TMD), and the intracellular domain (ICD), the molecular details of which are unclear. Here, we determined two serotonin-bound structures of the full-length 5-HT 3A R in distinct conformations at 3.32 Å and 3.89 Å resolutions, which we referred as State 1 and State 2, respectively. The new structures reveal the mechanism underlying channel activation. When compared to Apo-5-HT 3A R, serotonin-bound states underwent a large twisting motion in the ECD and TMD leading to the opening of a 165 Å long permeation pathway. Notably, this motion results in creation of lateral portals for ion permeation at the interface of the TMD and ICD. Combined with molecular dynamics simulations, these structures provide novel insights into conformational coupling across domains and functional modulation.

Serotonin receptor (5-HT 3A R) is the most common therapeutic target to manage the nausea and vomiting during cancer therapies and in the treatment of irritable bowel syndrome. Setrons, a class of competitive antagonists, cause functional inhibition of 5-HT 3A R in the gastrointestinal tract and brainstem, acting as effective anti-emetic agents. Despite their prevalent use, the molecular mechanisms underlying setron binding and inhibition of 5-HT 3A R are not fully understood. Here, we present the structure of granisetron-bound full-length 5-HT 3A R solved by single-particle cryo-electron microscopy to 2.92 Å resolution. The reconstruction reveals the orientation of granisetron in the orthosteric site with unambiguous density for interacting sidechains. Molecular dynamics simulations and electrophysiology confirm the granisetron binding orientation and the residues central for ligand recognition. Comparison of granisetron-bound 5-HT 3A R with the apo and serotonin-bound structures, reveals key insights into the mechanism underlying 5-HT 3A R inhibition.