Structural bases of inhibitory mechanism of CaV1.2 channel inhibitors.

The voltage-gated calcium channel CaV1.2 is essential for cardiac and vessel smooth muscle contractility and brain function. Accumulating evidence demonstrates that malfunctions of CaV1.2 are involved in brain and heart diseases. Pharmacological inhibition of CaV1.2 is therefore of therapeutic value. Here, we report cryo-EM structures of CaV1.2 in the absence or presence of the antirheumatic drug tetrandrine or antihypertensive drug benidipine. Tetrandrine acts as a pore blocker in a pocket composed of S6II, S6III, and S6IV helices and forms extensive hydrophobic interactions with CaV1.2. Our structure elucidates that benidipine is located in the DIII-DIV fenestration site. Its hydrophobic sidechain, phenylpiperidine, is positioned at the exterior of the pore domain and cradled within a hydrophobic pocket formed by S5DIII, S6DIII, and S6DIV helices, providing additional interactions to exert inhibitory effects on both L-type and T-type voltage gated calcium channels. These findings provide the structural foundation for the rational design and optimization of therapeutic inhibitors of voltage-gated calcium channels.

Structural insights into the human niacin receptor HCA2-Gi signalling complex.

The hydroxycarboxylic acid receptor 2 (HCA2) agonist niacin has been used as treatment for dyslipidemia for several decades albeit with skin flushing as a common side-effect in treated individuals. Extensive efforts have been made to identify HCA2 targeting lipid lowering agents with fewer adverse effects, despite little being known about the molecular basis of HCA2 mediated signalling. Here, we report the cryo-electron microscopy structure of the HCA2-Gi signalling complex with the potent agonist MK-6892, along with crystal structures of HCA2 in inactive state. These structures, together with comprehensive pharmacological analysis, reveal the ligand binding mode and activation and signalling mechanisms of HCA2. This study elucidates the structural determinants essential for HCA2 mediated signalling and provides insights into ligand discovery for HCA2 and related receptors.

Molecular insights into the gating mechanisms of voltage-gated calcium channel CaV2.3.

High-voltage-activated R-type CaV2.3 channel plays pivotal roles in many physiological activities and is implicated in epilepsy, convulsions, and other neurodevelopmental impairments. Here, we determine the high-resolution cryo-electron microscopy (cryo-EM) structure of human CaV2.3 in complex with the α2δ1 and ß1 subunits. The VSDII is stabilized in the resting state. Electrophysiological experiments elucidate that the VSDII is not required for channel activation, whereas the other VSDs are essential for channel opening. The intracellular gate is blocked by the W-helix. A pre-W-helix adjacent to the W-helix can significantly regulate closed-state inactivation (CSI) by modulating the association and dissociation of the W-helix with the gate. Electrostatic interactions formed between the negatively charged domain on S6II, which is exclusively conserved in the CaV2 family, and nearby regions at the alpha-interacting domain (AID) and S4-S5II helix are identified. Further functional analyses indicate that these interactions are critical for the open-state inactivation (OSI) of CaV2 channels.

Structural basis of autoinhibition of the human NHE3-CHP1 complex.

Sodium-proton exchanger 3 (NHE3/SLC9A3) located in the apical membrane of renal and gastrointestinal epithelia mediates salt and fluid absorption and regulates pH homeostasis. As an auxiliary regulatory factor of NHE proteins, calcineurin B homologous protein 1 (CHP1) facilitates NHE3 maturation, plasmalemmal expression, and pH sensitivity. Dysfunctions of NHE3 are associated with renal and digestive system disorders. Here, we report the cryo-electron microscopy structure of the human NHE3-CHP1 complex in its inward-facing conformation. We found that a cytosolic helix-loop-helix motif in NHE3 blocks the intracellular cavity formed between the core and dimerization domains, functioning as an autoinhibitory element and hindering substrate transport. Furthermore, two phosphatidylinositol molecules are found to bind to the peripheric juxtamembrane sides of the complex, function as anchors to stabilize the complex, and may thus enhance its transport activity.

Structural basis for modulation of human NaV1.3 by clinical drug and selective antagonist.

Voltage-gated sodium (NaV) channels play fundamental roles in initiating and propagating action potentials. NaV1.3 is involved in numerous physiological processes including neuronal development, hormone secretion and pain perception. Here we report structures of human NaV1.3/ß1/ß2 in complex with clinically-used drug bulleyaconitine A and selective antagonist ICA121431. Bulleyaconitine A is located around domain I-II fenestration, providing the detailed view of the site-2 neurotoxin binding site. It partially blocks ion path and expands the pore-lining helices, elucidating how the bulleyaconitine A reduces peak amplitude but improves channel open probability. In contrast, ICA121431 preferentially binds to activated domain IV voltage-sensor, consequently strengthens the Ile-Phe-Met motif binding to its receptor site, stabilizes the channel in inactivated state, revealing an allosterically inhibitory mechanism of NaV channels. Our results provide structural details of distinct small-molecular modulators binding sites, elucidate molecular mechanisms of their action on NaV channels and pave a way for subtype-selective therapeutic development.

Structure of human glycosylphosphatidylinositol transamidase.

Glycosylphosphatidylinositol (GPI) molecules are complex glycophospholipids and serve as membrane anchors for tethering many proteins to the cell surface. Attaching GPI to the protein in the endoplasmic reticulum (ER) is catalyzed by the transmembrane GPI transamidase (GPIT) complex, which is essential for maturation of the GPI-anchored proteins. The GPIT complex is known to be composed of five subunits: PIGK, PIGU, PIGT, PIGS and GPAA1. Here, we determined the structure of the human GPIT complex at a resolution of 3.1 Å using single-particle cryo-EM, elucidating its overall assembly. The PIGK subunit functions as the catalytic component, in which we identified a C206-H164-N58 triad that is critical for the transamination reaction. Transmembrane helices constitute a widely opened cleft, which is located underneath PIGK, serving as a GPI substrate-binding site. The ubiquitin E3 ligase RNF121 is visualized at the back of the complex and probably serves as a quality control factor for the GPIT complex.

Closed-state inactivation and pore-blocker modulation mechanisms of human CaV2.2.

N-type voltage-gated calcium (CaV) channels mediate Ca2+ influx at presynaptic terminals in response to action potentials and play vital roles in synaptogenesis, release of neurotransmitters, and nociceptive transmission. Here, we elucidate a cryo-electron microscopy (cryo-EM) structure of the human CaV2.2 complex in apo, ziconotide-bound, and two CaV2.2-specific pore blockers-bound states. The second voltage-sensing domain (VSD) is captured in a resting-state conformation, trapped by a phosphatidylinositol 4,5-bisphosphate (PIP2) molecule, which is distinct from the other three VSDs of CaV2.2, as well as activated VSDs observed in previous structures of CaV channels. This structure reveals the molecular basis for the unique inactivation process of CaV2.2 channels, in which the intracellular gate formed by S6 helices is closed and a W-helix from the domain II-III linker stabilizes closed-state inactivation. The structures of this inactivated, drug-bound complex lay a solid foundation for developing new state-dependent blockers for treatment of chronic pain.

Kainate receptor modulation by NETO2.

Glutamate-gated kainate receptors are ubiquitous in the central nervous system of vertebrates, mediate synaptic transmission at the postsynapse and modulate transmitter release at the presynapse1-7. In the brain, the trafficking, gating kinetics and pharmacology of kainate receptors are tightly regulated by neuropilin and tolloid-like (NETO) proteins8-11. Here we report cryo-electron microscopy structures of homotetrameric GluK2 in complex with NETO2 at inhibited and desensitized states, illustrating variable stoichiometry of GluK2-NETO2 complexes, with one or two NETO2 subunits associating with GluK2. We find that NETO2 accesses only two broad faces of kainate receptors, intermolecularly crosslinking the lower lobe of ATDA/C, the upper lobe of LBDB/D and the lower lobe of LBDA/C, illustrating how NETO2 regulates receptor-gating kinetics. The transmembrane helix of NETO2 is positioned proximal to the selectivity filter and competes with the amphiphilic H1 helix after M4 for interaction with an intracellular cap domain formed by the M1-M2 linkers of the receptor, revealing how rectification is regulated by NETO2.

Structural basis for distinct quality control mechanisms of GABAB receptor during evolution.

Cell surface trafficking of many G protein-coupled receptors is tightly regulated. Among them, the mandatory heterodimer GABAB receptor for the main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), is a model. In mammals, its cell surface trafficking is highly controlled by an endoplasmic reticulum retention signal in the C-terminal intracellular region of the GB1 subunit that is masked through a coiled-coil interaction with the GB2 subunit. Here, we investigate the molecular basis for the export of its homolog in Drosophila melanogaster that regulates the circadian rhythm and sleep. In contrast to mammals, the endoplasmic retention signal is carried by GB2, while GB1 reaches the cell surface alone. NMR analysis showed that the coiled-coil domain that controls GABAB heterodimer formation is structurally conserved between flies and mammals, despite specific features. These findings show the adaptation of a similar quality control system during evolution for maintaining the subunit composition of a functional heterodimeric receptor.

Crystal structure of dopamine receptor D4 bound to the subtype selective ligand, L745870.

Multiple subtypes of dopamine receptors within the GPCR superfamily regulate neurological processes through various downstream signaling pathways. A crucial question about the dopamine receptor family is what structural features determine the subtype-selectivity of potential drugs. Here, we report the 3.5-angstrom crystal structure of mouse dopamine receptor D4 (DRD4) complexed with a subtype-selective antagonist, L745870. Our structure reveals a secondary binding pocket extended from the orthosteric ligand-binding pocket to a DRD4-specific crevice located between transmembrane helices 2 and 3. Additional mutagenesis studies suggest that the antagonist L745870 prevents DRD4 activation by blocking the relative movement between transmembrane helices 2 and 3. These results expand our knowledge of the molecular basis for the physiological functions of DRD4 and assist new drug design.

Structure of YidC from Thermotoga maritima and its implications for YidC-mediated membrane protein insertion.

The evolutionarily conserved YidC/Oxa1/Alb3 family of proteins represents a unique membrane protein family that facilitates the insertion, folding, and assembly of a cohort of α-helical membrane proteins in all kingdoms of life, yet its underlying mechanisms remain elusive. We report the crystal structures of the full-length Thermotoga maritima YidC (TmYidC) and the TmYidC periplasmic domain (TmPD) at a resolution of 3.8 and 2.5 Å, respectively. The crystal structure of TmPD reveals a ß-supersandwich fold but with apparently shortened ß strands and different connectivity, as compared to the Escherichia coli YidC (EcYidC) periplasmic domain (EcPD). TmYidC in a detergent-solubilized state also adopts a monomeric form and its conserved core domain, which consists of 2 loosely associated α-helical bundles, assemble a fold similar to that of the other YidC homologues, yet distinct from that of the archaeal YidC-like DUF106 protein. Functional analysis using in vivo photo-crosslinking experiments demonstrates that Pf3 coat protein, a Sec-independent YidC substrate, exits to the lipid bilayer laterally via one of the 2 α-helical bundle interfaces: TM3-TM5. Engineered intramolecular disulfide bonds in TmYidC, in combination with complementation assays, suggest that significant rearrangement of the 2 α-helical bundles at the top of the hydrophilic groove is critical for TmYidC function. These experiments provide a more detailed mechanical insight into YidC-mediated membrane protein biogenesis.-Xin, Y., Zhao, Y., Zheng, J., Zhou, H., Zhang, X. C., Tian, C., Huang, Y. Structure of YidC from Thermotoga maritima and its implications for YidC-mediated membrane protein insertion.

Structure of the nonameric bacterial amyloid secretion channel.

Various strains of bacteria are able to produce a unique class of functional amyloids termed curli, which are critical for biofilm formation, host cell adhesion, and colonization of inert surfaces. Curli are secreted via the type VIII bacterial secretion system, and they share biochemical and structural characteristics with amyloid fibers that have been implicated in deleterious disease in humans. Here, we report the crystal structure of Escherichia coli CsgG, which is an essential lipoprotein component of the type VIII secretion system and which forms a secretion channel in the bacterial outer membrane for transporting curli subunits. CsgG forms a crown-shaped, symmetric nonameric channel that spans the outer membrane via a 36-strand ß-barrel, with each subunit contributing four ß-strands. This nonameric complex contains a central channel with a pore located at the middle. The eyelet of the pore is ∼12 Å in diameter and is lined with three stacked nine-residue rings consisting of Tyr-66, Asn-70, or Phe-71. Our structure-based functional studies suggest that Tyr-66 and Phe-71 residues function as gatekeepers for the selective secretion of curli subunits. Our study describes in detail, to our knowledge, the first core structure of the type VIII bacterial secretion machinery. Importantly, our structural analysis suggests that the curli subunits are secreted via CsgG across the bacterial outer membrane in an unfolded form.

Structural basis for lipopolysaccharide insertion in the bacterial outer membrane.

One of the fundamental properties of biological membranes is the asymmetric distribution of membrane lipids. In Gram-negative bacteria, the outer leaflet of the outer membrane is composed predominantly of lipopolysaccharides (LPS). The export of LPS requires seven essential lipopolysaccharide transport (Lpt) proteins to move LPS from the inner membrane, through the periplasm to the surface. Of the seven Lpt proteins, the LptD-LptE complex is responsible for inserting LPS into the external leaflet of the outer membrane. Here we report the crystal structure of the ∼110-kilodalton membrane protein complex LptD-LptE from Shigella flexneri at 2.4 Å resolution. The structure reveals an unprecedented two-protein plug-and-barrel architecture with LptE embedded into a 26-stranded ß-barrel formed by LptD. Importantly, the secondary structures of the first two ß-strands are distorted by two proline residues, weakening their interactions with neighbouring ß-strands and creating a potential portal on the barrel wall that could allow lateral diffusion of LPS into the outer membrane. The crystal structure of the LptD-LptE complex opens the door to new antibiotic strategies targeting the bacterial outer membrane.

Crystal structure of lipid phosphatase Escherichia coli phosphatidylglycerophosphate phosphatase B.

Membrane-integrated type II phosphatidic acid phosphatases (PAP2s) are important for numerous bacterial to human biological processes, including glucose transport, lipid metabolism, and signaling. Escherichia coli phosphatidylglycerol-phosphate phosphatase B (ecPgpB) catalyzes removing the terminal phosphate group from a lipid carrier, undecaprenyl pyrophosphate, and is essential for transport of many hydrophilic small molecules across the membrane. We determined the crystal structure of ecPgpB at a resolution of 3.2 Å. This structure shares a similar folding topology and a nearly identical active site with soluble PAP2 enzymes. However, the substrate binding mechanism appears to be fundamentally different from that in soluble PAP2 enzymes. In ecPgpB, the potential substrate entrance to the active site is located in a cleft formed by a V-shaped transmembrane helix pair, allowing lateral movement of the lipid substrate entering the active site from the membrane lipid bilayer. Activity assays of point mutations confirmed the importance of the catalytic residues and potential residues involved in phosphate binding. The structure also suggests an induced-fit mechanism for the substrate binding. The 3D structure of ecPgpB serves as a prototype to study eukaryotic PAP2 enzymes, including human glucose-6-phosphatase, a key enzyme in the homeostatic regulation of blood glucose concentrations.

Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A.

The major facilitator superfamily (MFS) is the largest family of secondary active transporters and is present in all life kingdoms. Detailed structural basis of the substrate transport and energy-coupling mechanisms of these proteins remain to be elucidated. YajR is a putative proton-driven MFS transporter found in many Gram-negative bacteria. Here we report the crystal structure of Escherichia coli YajR at 3.15 Å resolution in an outward-facing conformation. In addition to having the 12 canonical transmembrane helices, the YajR structure includes a unique 65-residue C-terminal domain which is independently stable. The structure is unique in illustrating the functional role of "sequence motif A." This highly conserved element is seen to stabilize the outward conformation of YajR and suggests a general mechanism for the conformational change between the inward and outward states of the MFS transporters.