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N-Methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors, which mediate most excitatory synaptic transmission in mammalian brains. Calcium permeation triggered by activation of NMDA receptors is the pivotal event for initiation of neuronal plasticity. Here, we show the crystal structure of the intact heterotetrameric GluN1-GluN2B NMDA receptor ion channel at 4 angstroms. The NMDA receptors are arranged as a dimer of GluN1-GluN2B heterodimers with the twofold symmetry axis running through the entire molecule composed of an amino terminal domain (ATD), a ligand-binding domain (LBD), and a transmembrane domain (TMD). The ATD and LBD are much more highly packed in the NMDA receptors than non-NMDA receptors, which may explain why ATD regulates ion channel activity in NMDA receptors but not in non-NMDA receptors.

A pivotal component of the calcium (Ca 2+ ) signaling toolbox in cells is the inositol 1,4,5-triphosphate (IP 3 ) receptor (IP 3 R), which mediates Ca 2+ release from the endoplasmic reticulum (ER), controlling cytoplasmic and organellar Ca 2+ concentrations. IP 3 Rs are co-activated by IP 3 and Ca 2+ , inhibited by Ca 2+ at high concentrations, and potentiated by ATP. However, the underlying molecular mechanisms are unclear. Here we report cryo-electron microscopy (cryo-EM) structures of human type-3 IP 3 R obtained from a single dataset in multiple gating conformations: IP 3 -ATP bound pre-active states with closed channels, IP 3 -ATP-Ca 2+ bound active state with an open channel, and IP 3 -ATP-Ca 2+ bound inactive state with a closed channel. The structures demonstrate how IP 3 -induced conformational changes prime the receptor for activation by Ca 2+ , how Ca 2+ binding leads to channel opening, and how ATP modulates the activity, providing insights into the long-sought questions regarding the molecular mechanism underpinning receptor activation and gating. IP 3 receptors are intracellular calcium channels involved in numerous signaling pathways. Here, the authors present the cryo-EM structures of type-3 IP 3 receptors in multiple gating conformations, including the active state revealing the molecular mechanism of the receptor activation.

Targeting NMDA receptors Ifenprodil, a phenylethanolamine first developed as an adrenergic antagonist and now widely used as an antihypertensive, also has a neuroprotective effect through receptors. A study of this interaction shows that the NMDA receptor subunits GluN1 and GluN2B form heterodimers that bind ifenprodil at the GluN1/GluN2B interface. Conformational mobility in the GluN2B amino-terminal domain is essential for ifenprodil-mediated inhibition of the NMDA receptors. These findings may be relevant to the design of therapeutics to target specific NMDA receptor subtypes for use in neurological disorders. Since it was discovered that the anti-hypertensive agent ifenprodil has neuroprotective activity through its effects on NMDA ( N -methyl-D-aspartate) receptors 1 , a determined effort has been made to understand the mechanism of action and to develop improved therapeutic compounds on the basis of this knowledge 2 , 3 , 4 . Neurotransmission mediated by NMDA receptors is essential for basic brain development and function 5 . These receptors form heteromeric ion channels and become activated after concurrent binding of glycine and glutamate to the GluN1 and GluN2 subunits, respectively. A functional hallmark of NMDA receptors is that their ion-channel activity is allosterically regulated by binding of small compounds to the amino-terminal domain (ATD) in a subtype-specific manner. Ifenprodil and related phenylethanolamine compounds, which specifically inhibit GluN1 and GluN2B NMDA receptors 6 , 7 , have been intensely studied for their potential use in the treatment of various neurological disorders and diseases, including depression, Alzheimer’s disease and Parkinson’s disease 2 , 4 . Despite considerable enthusiasm, mechanisms underlying the recognition of phenylethanolamines and ATD-mediated allosteric inhibition remain limited owing to a lack of structural information. Here we report that the GluN1 and GluN2B ATDs form a heterodimer and that phenylethanolamine binds at the interface between GluN1 and GluN2B, rather than within the GluN2B cleft. The crystal structure of the heterodimer formed between the GluN1b ATD from Xenopus laevis and the GluN2B ATD from Rattus norvegicus shows a highly distinct pattern of subunit arrangement that is different from the arrangements observed in homodimeric non-NMDA receptors and reveals the molecular determinants for phenylethanolamine binding. Restriction of domain movement in the bi-lobed structure of the GluN2B ATD, by engineering of an inter-subunit disulphide bond, markedly decreases sensitivity to ifenprodil, indicating that conformational freedom in the GluN2B ATD is essential for ifenprodil-mediated allosteric inhibition of NMDA receptors. These findings pave the way for improving the design of subtype-specific compounds with therapeutic value for neurological disorders and diseases.

The physiology of N -methyl- d -aspartate (NMDA) receptors is fundamental to brain development and function. NMDA receptors are ionotropic glutamate receptors that function as heterotetramers composed mainly of GluN1 and GluN2 subunits. Activation of NMDA receptors requires binding of neurotransmitter agonists to a ligand-binding domain (LBD) and structural rearrangement of an amino-terminal domain (ATD). Recent crystal structures of GluN1–GluN2B NMDA receptors bound to agonists and an allosteric inhibitor, ifenprodil, represent the allosterically inhibited state. However, how the ATD and LBD move to activate the NMDA receptor ion channel remains unclear. Here we applied X-ray crystallography, single-particle electron cryomicroscopy and electrophysiology to rat NMDA receptors to show that, in the absence of ifenprodil, the bi-lobed structure of GluN2 ATD adopts an open conformation accompanied by rearrangement of the GluN1–GluN2 ATD heterodimeric interface, altering subunit orientation in the ATD and LBD and forming an active receptor conformation that gates the ion channel. X-ray crystallography, single-particle electron cryomicroscopy and electrophysiology were used to study the conformational changes that take place during the activation and inhibition of a mammalian GluN1b–GluN2B N -methyl- d -aspartate receptor. Conformational alterations of NMDA receptor activation NMDA ( N -methyl- D -aspartate) receptors are ionotropic glutamate receptors that are involved in brain development and function, including learning and memory formation, and dysfunctional NMDA receptors are associated with various neurological diseases and disorders. These membrane proteins are heterotetramers, comprising two copies each of the GluN1 and GluN2 subunits, which bind glycine and L -glutamate, respectively. Hiro Furukawa and colleagues use X-ray crystallography, single-particle electron cryomicroscopy and electrophysiology to study the conformational changes that take place during the activation and inhibition of rat NMDA receptors. In the absence of the allosteric inhibitor ifenprodil, the bi-lobed structure of GluN2 amino-terminal domain adopts an open conformation accompanied by rearrangement of the GluN1–GluN2 amino-terminal domain heterodimeric interface, altering subunit orientation in the ligand-binding and amino-terminal domains to form an active receptor conformation that gates the ion channel.

N ‐methyl‐ D ‐aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino‐terminal domain (ATD) distinct from the L ‐glutamate‐binding domain. The molecular basis for the ATD‐mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc‐free and zinc‐bound states. The structures reveal the overall clamshell‐like architecture distinct from the non‐NMDA receptor ATDs and molecular determinants for the zinc‐binding site, ion‐binding sites, and the architecture of the putative phenylethanolamine‐binding site.

Volume-regulated anion channels (VRACs) mediate volume regulatory Cl - and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown stoichiometries. Homomeric LRRC8A and LRRC8D channels have a small pore, hexameric structure. However, these channels are either non-functional or exhibit abnormal regulation and pharmacology, limiting their utility for structure-function analyses. We circumvented these limitations by developing novel homomeric LRRC8 chimeric channels with functional properties consistent with those of native VRAC/LRRC8 channels. We demonstrate here that the LRRC8C-LRRC8A(IL1 25 ) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A has a heptameric structure like that of homologous pannexin channels. Unlike homomeric LRRC8A and LRRC8D channels, heptameric LRRC8C-LRRC8A(IL1 25 ) channels have a large-diameter pore similar to that estimated for native VRACs, exhibit normal DCPIB pharmacology, and have higher permeability to large organic anions. Lipid-like densities are located between LRRC8C-LRRC8A(IL1 25 ) subunits and occlude the channel pore. Our findings provide new insights into VRAC/LRRC8 channel structure and suggest that lipids may play important roles in channel gating and regulation.

In this study, we investigate the tangent bundle TM of an n-dimensional (pseudo-)Riemannian manifold M equipped with a Ricci-quarter symmetric metric connection ∇˜. Our primary goal is to establish the necessary and sufficient conditions for TM to exhibit characteristics of various solitons, specifically conformal Yamabe solitons, gradient conformal Yamabe solitons, conformal Ricci solitons, and gradient conformal Ricci solitons. We determine that for TM to be a conformal Yamabe soliton, the potential vector field must satisfy certain conditions when lifted vertically, horizontally, or completely from M to TM, alongside specific constraints on the conformal factor λ and the geometric properties of M. For gradient conformal Yamabe solitons, the conditions involve λ and the Hessian of the potential function. Similarly, for TM to be a conformal Ricci soliton, we identify conditions involving the lift of the potential vector field, the value of λ, and the curvature properties of M. For gradient conformal Ricci solitons, the criteria include the Hessian of the potential function and the Ricci curvature of M. These results enhance the understanding of the geometric properties of tangent bundles under Ricci-quarter symmetric metric connections and provide insights into their transition into various soliton states, contributing significantly to the field of differential geometry.

Protease-containing ABC transporters (PCATs) couple the energy of ATP hydrolysis to the processing and export of diverse cargo proteins across cell membranes to mediate antimicrobial resistance and quorum sensing. Here, we combine biochemical analysis, single particle cryoEM, and DEER spectroscopy in lipid bilayers along with computational analysis to illuminate the structural and energetic underpinnings of coupled cargo protein export. Our integrated investigation uncovers competitive interplay between nucleotides and cargo protein binding that ensures the latter’s orderly processing and subsequent transport. The energetics of cryoEM structures in lipid bilayers are congruent with the inferred mechanism from ATP turnover analysis and reveal a snapshot of a high-energy outward-facing conformation that provides an exit pathway into the lipid bilayer and/or the extracellular side. DEER investigation of the core ABC transporter suggests evolutionary tuning of the energetic landscape to fulfill the function of substrate processing prior to export. Protease containing ABC Transporters (PCAT) play a critical role in the translocation of polypeptides across membranes. Here, authors reveal the structural and energetic of the ATP-powered conformational cycle that enable this process.

Here we used cryo-electron microscopy (cryo-EM), double electron-electron resonance spectroscopy (DEER), and molecular dynamics (MD) simulations, to capture and characterize ATP- and substrate-bound inward-facing (IF) and occluded (OC) conformational states of the heterodimeric ATP binding cassette (ABC) multidrug exporter BmrCD in lipid nanodiscs. Supported by DEER analysis, the structures reveal that ATP-powered isomerization entails changes in the relative symmetry of the BmrC and BmrD subunits that propagates from the transmembrane domain to the nucleotide binding domain. The structures uncover asymmetric substrate and Mg 2+ binding which we hypothesize are required for triggering ATP hydrolysis preferentially in one of the nucleotide-binding sites. MD simulations demonstrate that multiple lipid molecules differentially bind the IF versus the OC conformation thus establishing that lipid interactions modulate BmrCD energy landscape. Our findings are framed in a model that highlights the role of asymmetric conformations in the ATP-coupled transport with general implications to the mechanism of ABC transporters. Multidrug resistance through active extrusion of molecules by transporters is a pressing clinical problem. Here, authors dissect the mechanism by which an ABC transporter from B. Subtilis binds and removes drugs by consuming the energy of ATP hydrolysis.

Leucine Rich Repeat Containing 8 (LRRC8) anion channels are emerging therapeutic targets, but their pharmacology is poorly developed. We employed a structurally defined homomeric channel chimera (8C-8A(IL1 25 )) and heteromeric LRRC8A/LRRC8C (8A/8C) channels to investigate the mechanism of action of two structurally distinct LRRC8 inhibitors: zafirlukast and pranlukast. Molecular dynamics simulations identified zafirlukast binding sites in 8C-8A(IL1 25 ) comprising the amino (N)-terminal domain (NTD) and inter-subunit fenestrae between transmembrane (TM) helices 1 and 2. Pranlukast also clusters in fenestrae albeit closer to the external pore. Patch clamp analysis revealed that mutations in NTD, TM1, and TM2 alter 8C-8A(IL1 25 ) and 8A/8C sensitivity to zafirlukast and pranlukast, suggesting a common mechanism. The association between voltage-dependent inactivation induced by mutations or low pH and inhibitor sensitivity suggests that drug inhibition involves disruption of protein-lipid interactions and destabilization of the pore. This may represent a common mechanism of LRRC8 channel inhibition by lipophilic drugs. Molecular modeling and functional mutagenesis analysis identify a novel mechanism of LRRC8 volume-regulated anion channel inhibition by structurally distinct lipophilic drugs