RESUMO
The heartbeat is initiated by voltage-gated sodium channel NaV1.5, which opens rapidly and triggers the cardiac action potential; however, the structural basis for pore opening remains unknown. Here, we blocked fast inactivation with a mutation and captured the elusive open-state structure. The fast inactivation gate moves away from its receptor, allowing asymmetric opening of pore-lining S6 segments, which bend and rotate at their intracellular ends to dilate the activation gate to â¼10 Å diameter. Molecular dynamics analyses predict physiological rates of Na+ conductance. The open-state pore blocker propafenone binds in a high-affinity pose, and drug-access pathways are revealed through the open activation gate and fenestrations. Comparison with mutagenesis results provides a structural map of arrhythmia mutations that target the activation and fast inactivation gates. These results give atomic-level insights into molecular events that underlie generation of the action potential, open-state drug block, and fast inactivation of cardiac sodium channels, which initiate the heartbeat.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Animais , Arritmias Cardíacas/genética , Microscopia Crioeletrônica , Células HEK293 , Frequência Cardíaca/efeitos dos fármacos , Humanos , Ativação do Canal Iônico , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação/genética , Miocárdio , Canal de Sódio Disparado por Voltagem NAV1.5/isolamento & purificação , Canal de Sódio Disparado por Voltagem NAV1.5/ultraestrutura , Propafenona/farmacologia , Conformação Proteica , Ratos , Sódio/metabolismo , Fatores de Tempo , Água/químicaRESUMO
Voltage-gated sodium channel Nav1.5 generates cardiac action potentials and initiates the heartbeat. Here, we report structures of NaV1.5 at 3.2-3.5 Å resolution. NaV1.5 is distinguished from other sodium channels by a unique glycosyl moiety and loss of disulfide-bonding capability at the NaVß subunit-interaction sites. The antiarrhythmic drug flecainide specifically targets the central cavity of the pore. The voltage sensors are partially activated, and the fast-inactivation gate is partially closed. Activation of the voltage sensor of Domain III allows binding of the isoleucine-phenylalanine-methionine (IFM) motif to the inactivation-gate receptor. Asp and Ala, in the selectivity motif DEKA, line the walls of the ion-selectivity filter, whereas Glu and Lys are in positions to accept and release Na+ ions via a charge-delocalization network. Arrhythmia mutation sites undergo large translocations during gating, providing a potential mechanism for pathogenic effects. Our results provide detailed insights into Nav1.5 structure, pharmacology, activation, inactivation, ion selectivity, and arrhythmias.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/ultraestrutura , Animais , Linhagem Celular , Células HEK293 , Coração/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp/métodos , Ratos , Sódio/metabolismo , Canais de Sódio/química , Relação Estrutura-Atividade , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/ultraestruturaRESUMO
Inorganic phosphate (Pi) is a fundamental macronutrient for all living organisms, the homeostasis of which is critical for numerous biological activities1-3. As the only known human Pi exporter to date, XPR1 has an indispensable role in cellular Pi homeostasis4,5. Dysfunction of XPR1 is associated with neurodegenerative disease6-8. However, the mechanisms underpinning XPR1-mediated Pi efflux and regulation by the intracellular inositol polyphosphate (InsPP) sensor SPX domain remain poorly understood. Here we present cryo-electron microscopy structures of human XPR1 in Pi-bound closed, open and InsP6-bound forms, revealing the structural basis for XPR1 gating and regulation by InsPPs. XPR1 consists of an N-terminal SPX domain, a dimer-formation core domain and a Pi transport domain. Within the transport domain, three basic clusters are responsible for Pi binding and transport, and a conserved W573 acts as a molecular switch for gating. In addition, the SPX domain binds to InsP6 and facilitates Pi efflux by liberating the C-terminal loop that limits Pi entry. This study provides a conceptual framework for the mechanistic understanding of Pi homeostasis by XPR1 homologues in fungi, plants and animals.
RESUMO
Vesicular monoamine transporter 2 (VMAT2) accumulates monoamines in presynaptic vesicles for storage and exocytotic release, and has a vital role in monoaminergic neurotransmission1-3. Dysfunction of monoaminergic systems causes many neurological and psychiatric disorders, including Parkinson's disease, hyperkinetic movement disorders and depression4-6. Suppressing VMAT2 with reserpine and tetrabenazine alleviates symptoms of hypertension and Huntington's disease7,8, respectively. Here we describe cryo-electron microscopy structures of human VMAT2 complexed with serotonin and three clinical drugs at 3.5-2.8 Å, demonstrating the structural basis for transport and inhibition. Reserpine and ketanserin occupy the substrate-binding pocket and lock VMAT2 in cytoplasm-facing and lumen-facing states, respectively, whereas tetrabenazine binds in a VMAT2-specific pocket and traps VMAT2 in an occluded state. The structures in three distinct states also reveal the structural basis of the VMAT2 transport cycle. Our study establishes a structural foundation for the mechanistic understanding of substrate recognition, transport, drug inhibition and pharmacology of VMAT2 while shedding light on the rational design of potential therapeutic agents.
Assuntos
Microscopia Crioeletrônica , Proteínas Vesiculares de Transporte de Monoamina , Humanos , Sítios de Ligação , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Ketanserina/química , Ketanserina/metabolismo , Ketanserina/farmacologia , Reserpina/química , Reserpina/metabolismo , Reserpina/farmacologia , Serotonina/química , Serotonina/metabolismo , Especificidade por Substrato , Tetrabenazina/química , Tetrabenazina/metabolismo , Tetrabenazina/farmacologia , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidores , Proteínas Vesiculares de Transporte de Monoamina/química , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/ultraestruturaRESUMO
Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.
Assuntos
Simulação por Computador , Genes Sintéticos/genética , Canais Iônicos/química , Canais Iônicos/genética , Modelos Moleculares , Biologia Sintética , Linhagem Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Condutividade Elétrica , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrazinas , Canais Iônicos/metabolismo , Transporte de Íons , Lipossomos/metabolismo , Técnicas de Patch-Clamp , Porinas/química , Porinas/genética , Porinas/metabolismo , Engenharia de Proteínas , Estrutura Secundária de Proteína , Solubilidade , Água/químicaRESUMO
The TRPV3 channel plays vital roles in skin physiology. Dysfunction of TRPV3 causes skin diseases, including Olmsted syndrome. However, the lack of potent and selective inhibitors impedes the validation of TRPV3 as a therapeutic target. In this study, we identified Trpvicin as a potent and subtype-selective inhibitor of TRPV3. Trpvicin exhibits pharmacological potential in the inhibition of itch and hair loss in mouse models. Cryogenic electron microscopy structures of TRPV3 and the pathogenic G573S mutant complexed with Trpvicin reveal detailed ligand-binding sites, suggesting that Trpvicin inhibits the TRPV3 channel by stabilizing it in a closed state. Our G573S mutant structures demonstrate that the mutation causes a dilated pore, generating constitutive opening activity. Trpvicin accesses additional binding sites inside the central cavity of the G573S mutant to remodel the channel symmetry and block the channel. Together, our results provide mechanistic insights into the inhibition of TRPV3 by Trpvicin and support TRPV3-related drug development.
Assuntos
Canais de Cátion TRPV , Camundongos , Animais , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/química , Mutação , Sítios de LigaçãoRESUMO
Potassium-sensitive hypokalaemic and normokalaemic periodic paralysis are inherited skeletal muscle diseases characterized by episodes of flaccid muscle weakness1,2. They are caused by single mutations in positively charged residues ('gating charges') in the S4 transmembrane segment of the voltage sensor of the voltage-gated sodium channel Nav1.4 or the calcium channel Cav1.11,2. Mutations of the outermost gating charges (R1 and R2) cause hypokalaemic periodic paralysis1,2 by creating a pathogenic gating pore in the voltage sensor through which cations leak in the resting state3,4. Mutations of the third gating charge (R3) cause normokalaemic periodic paralysis 5 owing to cation leak in both activated and inactivated states 6 . Here we present high-resolution structures of the model bacterial sodium channel NavAb with the analogous gating-charge mutations7,8, which have similar functional effects as in the human channels. The R2G and R3G mutations have no effect on the backbone structures of the voltage sensor, but they create an aqueous cavity near the hydrophobic constriction site that controls gating charge movement through the voltage sensor. The R3G mutation extends the extracellular aqueous cleft through the entire length of the activated voltage sensor, creating an aqueous path through the membrane. Conversely, molecular modelling shows that the R2G mutation creates a continuous aqueous path through the membrane only in the resting state. Crystal structures of NavAb(R2G) in complex with guanidinium define a potential drug target site. Molecular dynamics simulations illustrate the mechanism of Na+ permeation through the mutant gating pore in concert with conformational fluctuations of the gating charge R4. Our results reveal pathogenic mechanisms of periodic paralysis at the atomic level and suggest designs of drugs that may prevent ionic leak and provide symptomatic relief from hypokalaemic and normokalaemic periodic paralysis.
Assuntos
Ativação do Canal Iônico , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Paralisias Periódicas Familiares/metabolismo , Sítios de Ligação , Condutividade Elétrica , Guanidina/metabolismo , Humanos , Paralisia Periódica Hipopotassêmica/genética , Paralisia Periódica Hipopotassêmica/metabolismo , Ativação do Canal Iônico/genética , Simulação de Dinâmica Molecular , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Paralisias Periódicas Familiares/genética , Sódio/metabolismo , TermodinâmicaRESUMO
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.
Assuntos
Escherichia coli/enzimologia , Modelos Moleculares , Monoéster Fosfórico Hidrolases/química , Clonagem Molecular , Cristalografia por Raios X , Glucose-6-Fosfatase/química , Humanos , Conformação ProteicaRESUMO
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.
Assuntos
Motivos de Aminoácidos , Proteínas de Escherichia coli/química , Proteínas de Membrana Transportadoras/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Sítios de Ligação/genética , Transporte Biológico , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
YajR is an Escherichia coli transporter that belongs to the major facilitator superfamily. Unlike most MFS transporters, YajR contains a carboxyl terminal, cytosolic domain of 67 amino acid residues termed YAM domain. Although it is speculated that the function of this small soluble domain is to regulate the conformational change of the 12-helix transmembrane domain, its precise regulatory role remains unclear. Here, we report the crystal structure of the YAM domain at 1.07-Å resolution, along with its structure determined using nuclear magnetic resonance. Detailed analysis of the high resolution structure revealed a symmetrical dimer in which a belt of well-ordered poly-pentagonal water molecules is embedded. A mutagenesis experiment and a thermal stability assay were used to analyze the putative role of this dimerization in response to changes in halogen concentration.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Membrana Transportadoras/química , Sequência de Aminoácidos , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Multimerização Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Cloreto de Sódio/química , Água/químicaRESUMO
The prokaryotic 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the irreversible cleavage of the glycosidic bond in 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH), a process that plays a key role in several metabolic pathways. Its absence in all mammalian species has implicated this enzyme as a promising target for antimicrobial drug design. Here, we report the crystal structure of BmMTAN in complex with its product adenine at a resolution of 2.6 Å determined by single-wavelength anomalous dispersion method. 11 key residues were mutated for kinetic characterization. Mutations of Tyr134 and Met144 resulted in the largest overall increase in Km, whereas mutagenesis of residues Glu18, Glu145 and Asp168 completely abolished activity. Glu145 and Asp168 were identified as active site residues essential for catalysis. The catalytic mechanism and implications of this structure for broad-based antibiotic design are discussed.
Assuntos
Brucella melitensis/enzimologia , N-Glicosil Hidrolases/química , Adenina/química , Adenina/metabolismo , Brucella melitensis/química , Brucelose/microbiologia , Domínio Catalítico , Cristalografia por Raios X , Desoxiadenosinas/metabolismo , Cinética , Modelos Moleculares , N-Glicosil Hidrolases/metabolismo , Conformação Proteica , Especificidade por Substrato , Tionucleosídeos/metabolismoRESUMO
Voltage-gated sodium (NaV) channels mediate a plethora of electrical activities. NaV channels govern cellular excitability in response to depolarizing stimuli. Inactivation is an intrinsic property of NaV channels that regulates cellular excitability by controlling the channel availability. The fast inactivation, mediated by the Ile-Phe-Met (IFM) motif and the N-terminal helix (N-helix), has been well-characterized. However, the molecular mechanism underlying NaV channel slow inactivation remains elusive. Here, we demonstrate that the removal of the N-helix of NaVEh (NaVEhΔN) results in a slow-inactivated channel, and present cryo-EM structure of NaVEhΔN in a potential slow-inactivated state. The structure features a closed activation gate and a dilated selectivity filter (SF), indicating that the upper SF and the inner gate could serve as a gate for slow inactivation. In comparison to the NaVEh structure, NaVEhΔN undergoes marked conformational shifts on the intracellular side. Together, our results provide important mechanistic insights into NaV channel slow inactivation.
Assuntos
Microscopia Crioeletrônica , Ativação do Canal Iônico , Canais de Sódio Disparados por Voltagem , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/química , Humanos , Animais , Células HEK293 , Modelos MolecularesRESUMO
RNA uptake by cells is critical for RNA-mediated gene interference (RNAi) and RNA-based therapeutics. In Caenorhabditis elegans, RNAi is systemic as a result of SID-1-mediated double-stranded RNA (dsRNA) across cells. Despite the functional importance, the underlying mechanisms of dsRNA internalization by SID-1 remain elusive. Here we describe cryogenic electron microscopy structures of SID-1, SID-1-dsRNA complex and human SID-1 homologs SIDT1 and SIDT2, elucidating the structural basis of dsRNA recognition and import by SID-1. The homodimeric SID-1 homologs share conserved architecture, but only SID-1 possesses the molecular determinants within its extracellular domains for distinguishing dsRNA from single-stranded RNA and DNA. We show that the removal of the long intracellular loop between transmembrane helix 1 and 2 attenuates dsRNA uptake and systemic RNAi in vivo, suggesting a possible endocytic mechanism of SID-1-mediated dsRNA internalization. Our study provides mechanistic insights into dsRNA internalization by SID-1, which may facilitate the development of dsRNA applications based on SID-1.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Microscopia Crioeletrônica , RNA de Cadeia Dupla , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/química , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Humanos , Modelos Moleculares , Interferência de RNA , Proteínas de MembranaRESUMO
The sodium channel NaV1.6 is widely expressed in neurons of the central and peripheral nervous systems, which plays a critical role in regulating neuronal excitability. Dysfunction of NaV1.6 has been linked to epileptic encephalopathy, intellectual disability and movement disorders. Here we present cryo-EM structures of human NaV1.6/ß1/ß2 alone and complexed with a guanidinium neurotoxin 4,9-anhydro-tetrodotoxin (4,9-ah-TTX), revealing molecular mechanism of NaV1.6 inhibition by the blocker. The apo-form structure reveals two potential Na+ binding sites within the selectivity filter, suggesting a possible mechanism for Na+ selectivity and conductance. In the 4,9-ah-TTX bound structure, 4,9-ah-TTX binds to a pocket similar to the tetrodotoxin (TTX) binding site, which occupies the Na+ binding sites and completely blocks the channel. Molecular dynamics simulation results show that subtle conformational differences in the selectivity filter affect the affinity of TTX analogues. Taken together, our results provide important insights into NaV1.6 structure, ion conductance, and inhibition.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.6 , Bloqueadores dos Canais de Sódio , Tetrodotoxina , Humanos , Simulação de Dinâmica Molecular , Neurônios/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/análogos & derivados , Tetrodotoxina/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.6/químicaRESUMO
The sodium-activated Slo2.2 channel is abundantly expressed in the brain, playing a critical role in regulating neuronal excitability. The Na+-binding site and the underlying mechanisms of Na+-dependent activation remain unclear. Here, we present cryoelectron microscopy (cryo-EM) structures of human Slo2.2 in closed, open, and inhibitor-bound form at resolutions of 2.6-3.2 Å, revealing gating mechanisms of Slo2.2 regulation by cations and a potent inhibitor. The cytoplasmic gating ring domain of the closed Slo2.2 harbors multiple K+ and Zn2+ sites, which stabilize the channel in the closed conformation. The open Slo2.2 structure reveals at least two Na+-sensitive sites where Na+ binding induces expansion and rotation of the gating ring that opens the inner gate. Furthermore, a potent inhibitor wedges into a pocket formed by pore helix and S6 helix and blocks the pore. Together, our results provide a comprehensive structural framework for the investigation of Slo2.2 channel gating, Na+ sensation, and inhibition.
Assuntos
Canais de Potássio , Sódio , Humanos , Canais de Potássio/metabolismo , Microscopia Crioeletrônica , Canais de Potássio Ativados por Sódio , Sódio/metabolismoRESUMO
Voltage-gated sodium (NaV) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa, is the first reported plant-derived NaV channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV1.7. These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons.
Assuntos
Toxinas Biológicas , Urtica dioica , Austrália , Dor , Peptídeos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismoRESUMO
Glycoside hydrolase family 43 (GH43) consists of a variety of enzymes distributed widely in prokaryotes and eukaryotes. The mechanism by which GH43 enzymes hydrolyze oligosaccharides requires three essential acidic amino acid residues. However, one of them is thought to be missing in galactan ß-1,3-galactosidases from the GH43 family. Ct1,3Gal43A, from Clostridium thermocellum, is comprised of a GH43 domain, a CBM13 domain, and a dockerin domain and exhibits an unusual ability to hydrolyze ß-1,3-galactan in the presence of a ß-1,6 linked branch. Here, we present its crystal structure at 2.7 Å resolution and complex structures of the enzyme with several substrates and analogs. Two modes of substrate binding were observed at the ß site of the CtCBM13 domain, and one galactobiose molecule was found in an "L" shaped pocket of the CtGH43 domain, which appears large enough to accommodate two more galactose units. In addition, we found that mutating Glu112 to Gln or Ala eliminated the galactan hydrolysis activity of Ct1,3Gal43A while did not disrupt its ligand binding ability. Combining this results and the crystal structure we identified Glu112 in Ct1,3Gal43A as the 'missing' essential acidic residue in galactan ß-1,3-galactosidases. Structural information presented here also suggests a mechanism by which Ct1,3Gal43A bypasses ß-1,6 linked branches in the substrate and another mechanism by which the substrate is delivered 'in trans' from the CBM13 domain to the catalytic GH43 domain.
Assuntos
Clostridium thermocellum/química , Galactanos/química , Galactose/química , Ácido Glutâmico/química , Glicosídeo Hidrolases/química , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Clostridium thermocellum/enzimologia , Cristalografia por Raios X , Escherichia coli/genética , Ácido Glutâmico/genética , Glicosídeo Hidrolases/genética , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Especificidade por SubstratoRESUMO
Voltage-gated sodium (NaV) channels are responsible for the rapid rising-phase of action potentials in excitable cells. Over 1,000 mutations in NaV channels are associated with human diseases including epilepsy, periodic paralysis, arrhythmias and pain disorders. Natural toxins and clinically-used small-molecule drugs bind to NaV channels and modulate their functions. Recent advances from cryo-electron microscopy (cryo-EM) structures of NaV channels reveal invaluable insights into the architecture, activation, fast inactivation, electromechanical coupling, ligand modulation and pharmacology of eukaryotic NaV channels. These structural analyses not only demonstrate molecular mechanisms for NaV channel structure and function, but also provide atomic level templates for rational development of potential subtype-selective therapeutics. In this review, we summarize recent structural advances of eukaryotic NaV channels, highlighting the structural features of eukaryotic NaV channels as well as distinct modulation mechanisms by a wide range of modulators from natural toxins to synthetic small-molecules.
RESUMO
Voltage-gated sodium channel NaV1.7 plays essential roles in pain and odor perception. NaV1.7 variants cause pain disorders. Accordingly, NaV1.7 has elicited extensive attention in developing new analgesics. Here we present cryo-EM structures of human NaV1.7/ß1/ß2 complexed with inhibitors XEN907, TC-N1752 and NaV1.7-IN2, explaining specific binding sites and modulation mechanism for the pore blockers. These inhibitors bind in the central cavity blocking ion permeation, but engage different parts of the cavity wall. XEN907 directly causes α- to π-helix transition of DIV-S6 helix, which tightens the fast inactivation gate. TC-N1752 induces π-helix transition of DII-S6 helix mediated by a conserved asparagine on DIII-S6, which closes the activation gate. NaV1.7-IN2 serves as a pore blocker without causing conformational change. Electrophysiological results demonstrate that XEN907 and TC-N1752 stabilize NaV1.7 in inactivated state and delay the recovery from inactivation. Our results provide structural framework for NaV1.7 modulation by pore blockers, and important implications for developing subtype-selective analgesics.
Assuntos
Dor , Humanos , Sítios de LigaçãoRESUMO
Voltage-gated sodium (NaV) channels initiate action potentials. Fast inactivation of NaV channels, mediated by an Ile-Phe-Met motif, is crucial for preventing hyperexcitability and regulating firing frequency. Here we present cryo-electron microscopy structure of NaVEh from the coccolithophore Emiliania huxleyi, which reveals an unexpected molecular gating mechanism for NaV channel fast inactivation independent of the Ile-Phe-Met motif. An N-terminal helix of NaVEh plugs into the open activation gate and blocks it. The binding pose of the helix is stabilized by multiple electrostatic interactions. Deletion of the helix or mutations blocking the electrostatic interactions completely abolished the fast inactivation. These strong interactions enable rapid inactivation, but also delay recovery from fast inactivation, which is ~160-fold slower than human NaV channels. Together, our results provide mechanistic insights into fast inactivation of NaVEh that fundamentally differs from the conventional local allosteric inhibition, revealing both surprising structural diversity and functional conservation of ion channel inactivation.