RESUMO
ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.
Assuntos
Channelrhodopsins/química , Channelrhodopsins/metabolismo , Ativação do Canal Iônico , Animais , Channelrhodopsins/ultraestrutura , Microscopia Crioeletrônica , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Modelos Moleculares , Optogenética , Filogenia , Ratos Sprague-Dawley , Bases de Schiff/química , Células Sf9 , Relação Estrutura-AtividadeRESUMO
Type VI CRISPR-Cas13 effector enzymes catalyze RNA-guided RNA cleavage and have been harnessed for various technologies, such as RNA detection, targeting, and editing. Recent studies identified Cas13bt3 (also known as Cas13X.1) as a miniature Cas13 enzyme, which can be used for knockdown and editing of target transcripts in mammalian cells. However, the action mechanism of the compact Cas13bt3 remains unknown. Here, we report the structures of the Cas13bt3-guide RNA complex and the Cas13bt3-guide RNA-target RNA complex. The structures revealed how Cas13bt3 recognizes the guide RNA and its target RNA and provided insights into the activation mechanism of Cas13bt3, which is distinct from those of the other Cas13a/d enzymes. Furthermore, we rationally engineered enhanced Cas13bt3 variants and ultracompact RNA base editors. Overall, this study improves our mechanistic understanding of the CRISPR-Cas13 enzymes and paves the way for the development of efficient Cas13-mediated transcriptome modulation technologies.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Cinetoplastídeos , Animais , Edição de Genes , Mamíferos/genética , RNA/genética , RNA Guia de Cinetoplastídeos/genética , TranscriptomaRESUMO
Endogenous parathyroid hormone (PTH) and PTH-related peptide (PTHrP) bind to the parathyroid hormone receptor 1 (PTH1R) and activate the stimulatory G-protein (Gs) signaling pathway. Intriguingly, the two ligands have distinct signaling and physiological properties: PTH evokes prolonged Gs activation, whereas PTHrP evokes transient Gs activation with reduced bone-resorption effects. The distinct molecular actions are ascribed to the differences in ligand recognition and dissociation kinetics. Here, we report cryoelectron microscopic structures of six forms of the human PTH1R-Gs complex in the presence of PTH or PTHrP at resolutions of 2.8 -4.1 Å. A comparison of the PTH-bound and PTHrP-bound structures reveals distinct ligand-receptor interactions underlying the ligand affinity and selectivity. Furthermore, five distinct PTH-bound structures, combined with computational analyses, provide insights into the unique and complex process of ligand dissociation from the receptor and shed light on the distinct durations of signaling induced by PTH and PTHrP.
Assuntos
Proteína Relacionada ao Hormônio Paratireóideo , Receptor Tipo 1 de Hormônio Paratireóideo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Hormônio Paratireóideo/química , Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/farmacologia , Proteína Relacionada ao Hormônio Paratireóideo/química , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismoRESUMO
RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) and recognizes double-stranded DNA targets with a short TN PAM. Here, we report the cryo-electron microscopy structures of the Cas12c2-guide RNA binary complex and the Cas12c2-guide RNA-target DNA ternary complex. The structures revealed that the crRNA and tracrRNA form an unexpected X-junction architecture, and that Cas12c2 recognizes a single T nucleotide in the PAM through specific hydrogen-bonding interactions with two arginine residues. Furthermore, our biochemical analyses indicated that Cas12c2 processes its precursor crRNA to a mature crRNA using the RuvC catalytic site through a unique mechanism. Collectively, our findings improve the mechanistic understanding of diverse type V CRISPR-Cas effectors.
Assuntos
Proteínas Associadas a CRISPR , RNA Guia de Cinetoplastídeos , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Microscopia Crioeletrônica , DNA/genética , RNA Guia de Cinetoplastídeos/metabolismo , Ribonucleases/metabolismoRESUMO
G protein-coupled receptors (GPCRs) generally accommodate specific ligands in the orthosteric-binding pockets. Ligand binding triggers a receptor allosteric conformational change that leads to the activation of intracellular transducers, G proteins and ß-arrestins. Because these signals often induce adverse effects, the selective activation mechanism for each transducer must be elucidated. Thus, many orthosteric-biased agonists have been developed, and intracellular-biased agonists have recently attracted broad interest. These agonists bind within the receptor intracellular cavity and preferentially tune the specific signalling pathway over other signalling pathways, without allosteric rearrangement of the receptor from the extracellular side1-3. However, only antagonist-bound structures are currently available1,4-6, and there is no evidence to support that biased agonist binding occurs within the intracellular cavity. This limits the comprehension of intracellular-biased agonism and potential drug development. Here we report the cryogenic electron microscopy structure of a complex of Gs and the human parathyroid hormone type 1 receptor (PTH1R) bound to a PTH1R agonist, PCO371. PCO371 binds within an intracellular pocket of PTH1R and directly interacts with Gs. The PCO371-binding mode rearranges the intracellular region towards the active conformation without extracellularly induced allosteric signal propagation. PCO371 stabilizes the significantly outward-bent conformation of transmembrane helix 6, which facilitates binding to G proteins rather than ß-arrestins. Furthermore, PCO371 binds within the highly conserved intracellular pocket, activating 7 out of the 15 class B1 GPCRs. Our study identifies a new and conserved intracellular agonist-binding pocket and provides evidence of a biased signalling mechanism that targets the receptor-transducer interface.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP , Imidazolidinas , Receptores Acoplados a Proteínas G , Humanos , Regulação Alostérica , beta-Arrestinas/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Desenvolvimento de Medicamentos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Imidazolidinas/química , Imidazolidinas/farmacologia , Ligantes , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/classificação , Receptores Acoplados a Proteínas G/ultraestrutura , Transdução de SinaisRESUMO
The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors.
Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Elementos de DNA Transponíveis , Deinococcus , Endodesoxirribonucleases , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA/química , DNA/genética , DNA/metabolismo , DNA/ultraestrutura , Elementos de DNA Transponíveis/genética , RNA Guia de Sistemas CRISPR-Cas/química , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , RNA Guia de Sistemas CRISPR-Cas/ultraestrutura , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/ultraestrutura , Deinococcus/enzimologia , Deinococcus/genética , Especificidade por SubstratoRESUMO
The cytoplasmic polyamine maintains cellular homeostasis by chelating toxic metal cations, regulating transcriptional activity, and protecting DNA. ATP13A2 was identified as a lysosomal polyamine exporter responsible for polyamine release into the cytosol, and its dysfunction is associated with Alzheimer's disease and other neural degradation diseases. ATP13A2 belongs to the P5 subfamily of the P-type ATPase family, but its mechanisms remain unknown. Here, we report the cryoelectron microscopy (cryo-EM) structures of human ATP13A2 under four different conditions, revealing the structural coupling between the polyamine binding and the dephosphorylation. Polyamine is bound at the luminal tunnel and recognized through numerous electrostatic and π-cation interactions, explaining its broad specificity. The unique N-terminal domain is anchored to the lipid membrane to stabilize the E2P conformation, thereby accelerating the E1P-to-E2P transition. These findings reveal the distinct mechanism of P5B ATPases, thereby paving the way for neuroprotective therapy by activating ATP13A2.
Assuntos
Adenosina Trifosfatases/química , Lipídeos/química , Poliaminas/química , ATPases Translocadoras de Prótons/química , Sítios de Ligação , Microscopia Crioeletrônica , Citosol/metabolismo , Células HEK293 , Homeostase , Humanos , Lipídeos de Membrana/química , Micelas , Conformação Molecular , Fosforilação , Conformação ProteicaRESUMO
RNA-guided DNA endonucleases derived from CRISPR-Cas adaptive immune systems are widely used as powerful genome-engineering tools. Among the diverse CRISPR-Cas nucleases, the type V-F Cas12f (also known as Cas14) proteins are exceptionally compact and associate with a guide RNA to cleave single- and double-stranded DNA targets. Here, we report the cryo-electron microscopy structure of Cas12f1 (also known as Cas14a) in complex with a guide RNA and its target DNA. Unexpectedly, the structure revealed that two Cas12f1 molecules assemble with the single guide RNA to recognize the double-stranded DNA target. Each Cas12f1 protomer adopts a different conformation and plays distinct roles in nucleic acid recognition and DNA cleavage, thereby explaining how the miniature Cas12f1 enzyme achieves RNA-guided DNA cleavage as an "asymmetric homodimer." Our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and provide a framework for the development of compact genome-engineering tools critical for therapeutic genome editing.
Assuntos
Proteínas Associadas a CRISPR/ultraestrutura , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/ultraestrutura , Edição de Genes , RNA Guia de Cinetoplastídeos/ultraestrutura , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Microscopia Crioeletrônica , DNA/genética , DNA/metabolismo , Modelos Moleculares , Motivos de Nucleotídeos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
In flies, Argonaute2 (Ago2) and small interfering RNA (siRNA) form an RNA-induced silencing complex to repress viral transcripts1. The RNase III enzyme Dicer-2 associates with its partner protein R2D2 and cleaves long double-stranded RNAs to produce 21-nucleotide siRNA duplexes, which are then loaded into Ago2 in a defined orientation2-5. Here we report cryo-electron microscopy structures of the Dicer-2-R2D2 and Dicer-2-R2D2-siRNA complexes. R2D2 interacts with the helicase domain and the central linker of Dicer-2 to inhibit the promiscuous processing of microRNA precursors by Dicer-2. Notably, our structure represents the strand-selection state in the siRNA-loading process, and reveals that R2D2 asymmetrically recognizes the end of the siRNA duplex with the higher base-pairing stability, and the other end is exposed to the solvent and is accessible by Ago2. Our findings explain how R2D2 senses the thermodynamic asymmetry of the siRNA and facilitates the siRNA loading into Ago2 in a defined orientation, thereby determining which strand of the siRNA duplex is used by Ago2 as the guide strand for target silencing.
Assuntos
Microscopia Crioeletrônica , Proteínas de Drosophila , RNA Helicases , RNA de Cadeia Dupla , RNA Interferente Pequeno , Proteínas de Ligação a RNA , Ribonuclease III , Animais , Proteínas Argonautas/metabolismo , Pareamento de Bases , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , MicroRNAs/metabolismo , Multimerização Proteica , RNA Helicases/química , RNA Helicases/metabolismo , RNA Helicases/ultraestrutura , Interferência de RNA , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/ultraestrutura , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/ultraestrutura , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Complexo de Inativação Induzido por RNA/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Ribonuclease III/ultraestruturaRESUMO
Modulation of voltage-gated potassium (Kv) channels by auxiliary subunits is central to the physiological function of channels in the brain and heart1,2. Native Kv4 tetrameric channels form macromolecular ternary complexes with two auxiliary ß-subunits-intracellular Kv channel-interacting proteins (KChIPs) and transmembrane dipeptidyl peptidase-related proteins (DPPs)-to evoke rapidly activating and inactivating A-type currents, which prevent the backpropagation of action potentials1-5. However, the modulatory mechanisms of Kv4 channel complexes remain largely unknown. Here we report cryo-electron microscopy structures of the Kv4.2-DPP6S-KChIP1 dodecamer complex, the Kv4.2-KChIP1 and Kv4.2-DPP6S octamer complexes, and Kv4.2 alone. The structure of the Kv4.2-KChIP1 complex reveals that the intracellular N terminus of Kv4.2 interacts with its C terminus that extends from the S6 gating helix of the neighbouring Kv4.2 subunit. KChIP1 captures both the N and the C terminus of Kv4.2. In consequence, KChIP1 would prevent N-type inactivation and stabilize the S6 conformation to modulate gating of the S6 helices within the tetramer. By contrast, unlike the reported auxiliary subunits of voltage-gated channel complexes, DPP6S interacts with the S1 and S2 helices of the Kv4.2 voltage-sensing domain, which suggests that DPP6S stabilizes the conformation of the S1-S2 helices. DPP6S may therefore accelerate the voltage-dependent movement of the S4 helices. KChIP1 and DPP6S do not directly interact with each other in the Kv4.2-KChIP1-DPP6S ternary complex. Thus, our data suggest that two distinct modes of modulation contribute in an additive manner to evoke A-type currents from the native Kv4 macromolecular complex.
Assuntos
Microscopia Crioeletrônica , Ativação do Canal Iônico , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Canais de Potássio Shal/química , Canais de Potássio Shal/metabolismo , Animais , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Feminino , Humanos , Proteínas Interatuantes com Canais de Kv/química , Proteínas Interatuantes com Canais de Kv/metabolismo , Modelos Moleculares , Complexos Multiproteicos/genética , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Oócitos/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Ligação Proteica , Canais de Potássio Shal/genética , Xenopus laevisRESUMO
MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.
Assuntos
Antiporters/química , Proteínas de Bactérias/química , Ativação do Canal Iônico/fisiologia , Antiporters/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação/efeitos dos fármacos , Transporte Biológico , Microscopia Crioeletrônica , Cristalografia por Raios X , Citoplasma/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Magnésio/metabolismo , Magnésio/farmacologia , Modelos Moleculares , Domínios Proteicos/efeitos dos fármacos , Domínios Proteicos/fisiologia , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Thermus thermophilus/metabolismoRESUMO
In both prokaryotes and eukaryotes, multidrug and toxic-compound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e., their drug-efflux activity is coupled to, and powered by, the uptake of ions down a preexisting transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na+-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at atomic resolution, i.e. better than 3 Å. Reinterpretation of existing crystallographic data and molecular dynamics simulations indeed reveal an occupied Na+-binding site in the N-terminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K+, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na+-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na+ in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H+-dependent activity likely stem either from a site elsewhere in the structure, or from H+ displacing Na+ under certain laboratory conditions, as has been noted for other Na+-driven transport systems.
Assuntos
Antiporters/química , Proteínas de Transporte de Cátions Orgânicos/química , Conformação Proteica/efeitos dos fármacos , Sódio/química , Antibacterianos/efeitos adversos , Antibacterianos/farmacologia , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Antiporters/ultraestrutura , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Íons/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas de Transporte de Cátions Orgânicos/ultraestrutura , Células Procarióticas/química , Células Procarióticas/ultraestrutura , Domínios Proteicos/efeitos dos fármacosRESUMO
In both prokaryotes and eukaryotes, multidrug and toxic-compound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e. their drug-efflux activity is coupled to, and powered by, the uptake of ions down a pre-existing transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na+-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at resolutions better than 3 Å. Analysis of available crystallographic data and molecular dynamics simulations indeed reveal an occupied Na+-binding site in the N-terminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K+, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na+-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na+ in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H+-dependent activity stem either from a site elsewhere in the structure, or from H+ displacing Na+ under certain laboratory conditions, as has been noted for other Na+-driven transport systems.
RESUMO
The variety of taste sensations, including sweet, umami, bitter, sour, and salty, arises from diverse taste cells, each of which expresses specific taste sensor molecules and associated components for downstream signal transduction cascades. Recent years have witnessed major advances in our understanding of the molecular mechanisms underlying transduction of basic tastes in taste buds, including the identification of the bona fide sour sensor H+ channel OTOP1, and elucidation of transduction of the amiloride-sensitive component of salty taste (the taste of sodium) and the TAS1R-independent component of sweet taste (the taste of sugar). Studies have also discovered an unconventional chemical synapse termed "channel synapse" which employs an action potential-activated CALHM1/3 ion channel instead of exocytosis of synaptic vesicles as the conduit for neurotransmitter release that links taste cells to afferent neurons. New images of the channel synapse and determinations of the structures of CALHM channels have provided structural and functional insights into this unique synapse. In this review, we discuss the current view of taste transduction and neurotransmission with emphasis on recent advances in the field.
Assuntos
Sinapses/classificação , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Papilas Gustativas/fisiologia , Paladar/fisiologia , Animais , HumanosRESUMO
Secretin is a gastrointestinal hormone that exerts multiple physiological functions via activation of the secretin receptor (SECR). SECR belongs to the class B G-protein-coupled receptors and is involved in various processes, such as regulation of the pH of the duodenal content, food intake, and water homeostasis. Here, we report a cryo-electron microscopy structure of human SECR bound to secretin and an engineered Gs heterotrimer. The structure revealed the basic architecture of SECR and the secretin binding mode. A structural comparison of the SECR and PAC1R transmembrane domains revealed that transmembrane helices 1 and 2 play a prominent role in secretin recognition. Moreover, the extracellular domain of SECR is perpendicular to the TMD, unlike that of PAC1R. This comparison revealed the diverged peptide recognition mechanisms of these receptors, which belong to the same subgroup. Our structural information will facilitate drug discovery research for clinical applications.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/química , Receptores Acoplados a Proteínas G/química , Receptores dos Hormônios Gastrointestinais/química , Microscopia Crioeletrônica , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Engenharia de Proteínas , Secretina/químicaRESUMO
Vitamin C plays important roles as a cofactor in many enzymatic reactions and as an antioxidant against oxidative stress. As some mammals including humans cannot synthesize vitamin C de novo from glucose, its uptake from dietary sources is essential, and is mediated by the sodium-dependent vitamin C transporter 1 (SVCT1). Despite its physiological significance in maintaining vitamin C homeostasis, the structural basis of the substrate transport mechanism remained unclear. Here, we report the cryo-EM structures of human SVCT1 in different states at 2.5-3.5 Å resolutions. The binding manner of vitamin C together with two sodium ions reveals the counter ion-dependent substrate recognition mechanism. Furthermore, comparisons of the inward-open and occluded structures support a transport mechanism combining elevator and distinct rotational motions. Our results demonstrate the molecular mechanism of vitamin C transport with its underlying conformational cycle, potentially leading to future industrial and medical applications.
Assuntos
Ácido Ascórbico , Microscopia Crioeletrônica , Transportadores de Sódio Acoplados à Vitamina C , Humanos , Transportadores de Sódio Acoplados à Vitamina C/metabolismo , Transportadores de Sódio Acoplados à Vitamina C/química , Transportadores de Sódio Acoplados à Vitamina C/genética , Ácido Ascórbico/metabolismo , Ácido Ascórbico/química , Transporte Biológico , Sódio/metabolismo , Modelos Moleculares , Multimerização Proteica , Ligação Proteica , Células HEK293 , Conformação ProteicaRESUMO
RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR-Cas type V-M) is highly compact and has a unique RuvC active site. Although the non-canonical RuvC triad does not permit dsDNA cleavage, Cas12m2 still protects against invading MGEs through transcriptional silencing by strong DNA binding. However, the molecular mechanism of RNA-guided genome inactivation by Cas12m2 remains unknown. Here we report cryo-electron microscopy structures of two states of Cas12m2-CRISPR RNA (crRNA)-target DNA ternary complexes and the Cas12m2-crRNA binary complex, revealing structural dynamics during crRNA-target DNA heteroduplex formation. The structures indicate that the non-target DNA strand is tightly bound to a unique arginine-rich cluster in the recognition (REC) domains and the non-canonical active site in the RuvC domain, ensuring strong DNA-binding affinity of Cas12m2. Furthermore, a structural comparison of Cas12m2 with TnpB, a putative ancestor of Cas12 enzymes, suggests that the interaction of the characteristic coiled-coil REC2 insertion with the protospacer-adjacent motif-distal region of the heteroduplex is crucial for Cas12m2 to engage in adaptive immunity. Collectively, our findings improve mechanistic understanding of diverse type V CRISPR-Cas effectors and provide insights into the evolution of TnpB to Cas12 enzymes.
Assuntos
Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Microscopia Crioeletrônica , Bactérias/metabolismo , RNA/metabolismo , DNA/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismoRESUMO
Outer hair cell elecromotility, driven by prestin, is essential for mammalian cochlear amplification. Here, we report the cryo-EM structures of thermostabilized prestin (PresTS), complexed with chloride, sulfate, or salicylate at 3.52-3.63 Å resolutions. The central positively-charged cavity allows flexible binding of various anion species, which likely accounts for the known distinct modulations of nonlinear capacitance (NLC) by different anions. Comparisons of these PresTS structures with recent prestin structures suggest rigid-body movement between the core and gate domains, and provide mechanistic insights into prestin inhibition by salicylate. Mutations at the dimeric interface severely diminished NLC, suggesting that stabilization of the gate domain facilitates core domain movement, thereby contributing to the expression of NLC. These findings advance our understanding of the molecular mechanism underlying mammalian cochlear amplification.
Assuntos
Proteínas de Transporte de Ânions , Cloretos , Animais , Proteínas de Transporte de Ânions/metabolismo , Cloretos/metabolismo , Microscopia Crioeletrônica , Células Ciliadas Auditivas Externas/metabolismo , Ânions/metabolismo , Salicilatos , Sulfatos/metabolismo , Mamíferos/metabolismoRESUMO
Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.
Assuntos
Transportador 2 de Aminoácido Excitatório/química , Ácido Glutâmico , Animais , Sítios de Ligação , Encéfalo/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 3 de Aminoácido Excitatório/genética , Transportador 3 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Mamíferos/metabolismo , Neuroglia/metabolismoRESUMO
In the light reaction of plant photosynthesis, modulation of electron transport chain reactions is important to maintain the efficiency of photosynthesis under a broad range of light intensities. VCCN1 was recently identified as a voltage-gated chloride channel residing in the thylakoid membrane, where it plays a key role in photoreaction tuning to avoid the generation of reactive oxygen species (ROS). Here, we present the cryo-EM structures of Malus domestica VCCN1 (MdVCCN1) in nanodiscs and detergent at 2.7 Å and 3.0 Å resolutions, respectively, and the structure-based electrophysiological analyses. VCCN1 structurally resembles its animal homolog, bestrophin, a Ca2+-gated anion channel. However, unlike bestrophin channels, VCCN1 lacks the Ca2+-binding motif but instead contains an N-terminal charged helix that is anchored to the lipid membrane through an additional amphipathic helix. Electrophysiological experiments demonstrate that these structural elements are essential for the channel activity, thus revealing the distinct activation mechanism of VCCN1.