RESUMEN
The type III-E CRISPR-Cas effector Cas7-11, with dual RNase activities for precursor CRISPR RNA (pre-crRNA) processing and crRNA-guided target RNA cleavage, is a new platform for bacterial and mammalian RNA targeting. We report the 2.5-Å resolution cryoelectron microscopy structure of Cas7-11 in complex with a crRNA and its target RNA. Cas7-11 adopts a modular architecture comprising seven domains (Cas7.1-Cas7.4, Cas11, INS, and CTE) and four interdomain linkers. The crRNA 5' tag is recognized and processed by Cas7.1, whereas the crRNA spacer hybridizes with the target RNA. Consistent with our biochemical data, the catalytic residues for programmable cleavage in Cas7.2 and Cas7.3 neighbor the scissile phosphates before the flipped-out fourth and tenth nucleotides in the target RNA, respectively. Using structural insights, we rationally engineered a compact Cas7-11 variant (Cas7-11S) for single-vector AAV packaging for transcript knockdown in human cells, enabling in vivo Cas7-11 applications.
Asunto(s)
Proteínas Asociadas a CRISPR , Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas , Microscopía por Crioelectrón , Humanos , Precursores del ARN , ARN Bacteriano/química , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genéticaRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Kinetoplastida , Animales , Edición Génica , Mamíferos/genética , ARN/genética , ARN Guía de Kinetoplastida/genética , TranscriptomaRESUMEN
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.
Asunto(s)
Proteína Relacionada con la Hormona Paratiroidea , Receptor de Hormona Paratiroídea Tipo 1 , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Ligandos , Hormona Paratiroidea/química , Hormona Paratiroidea/metabolismo , Hormona Paratiroidea/farmacología , Proteína Relacionada con la Hormona Paratiroidea/química , Proteína Relacionada con la Hormona Paratiroidea/genética , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/genética , Receptor de Hormona Paratiroídea Tipo 1/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Asociadas a CRISPR , ARN Guía de Kinetoplastida , Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Microscopía por Crioelectrón , ADN/genética , ARN Guía de Kinetoplastida/metabolismo , Ribonucleasas/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/química , Lípidos/química , Poliaminas/química , ATPasas de Translocación de Protón/química , Sitios de Unión , Microscopía por Crioelectrón , Citosol/metabolismo , Células HEK293 , Homeostasis , Humanos , Lípidos de la Membrana/química , Micelas , Conformación Molecular , Fosforilación , Conformación ProteicaRESUMEN
The ß3-adrenergic receptor (ß3AR) is predominantly expressed in adipose tissue and urinary bladder and has emerged as an attractive drug target for the treatment of type 2 diabetes, obesity, and overactive bladder (OAB). Here, we report the cryogenic electron microscopy structure of the ß3AR-Gs signaling complex with the selective agonist mirabegron, a first-in-class drug for OAB. Comparison of this structure with the previously reported ß1AR and ß2AR structures reveals a receptor activation mechanism upon mirabegron binding to the orthosteric site. Notably, the narrower exosite in ß3AR creates a perpendicular pocket for mirabegron. Mutational analyses suggest that a combination of both the exosite shape and the amino-acid-residue substitutions defines the drug selectivity of the ßAR agonists. Our findings provide a molecular basis for ßAR subtype selectivity, allowing the design of more-selective agents with fewer adverse effects.
Asunto(s)
Acetanilidas/química , Agonistas de Receptores Adrenérgicos beta 3/química , Receptores Adrenérgicos beta 3/química , Receptores Adrenérgicos beta 3/metabolismo , Tiazoles/química , Acetanilidas/metabolismo , Agonistas de Receptores Adrenérgicos beta 3/metabolismo , Animales , Sitios de Unión , Microscopía por Crioelectrón , Perros , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Receptores Adrenérgicos beta 3/genética , Tiazoles/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Asociadas a CRISPR/ultraestructura , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/ultraestructura , Edición Génica , ARN Guía de Kinetoplastida/ultraestructura , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Microscopía por Crioelectrón , ADN/genética , ADN/metabolismo , Modelos Moleculares , Motivos de Nucleótidos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Subunidades de Proteína , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Microscopía por Crioelectrón , Proteínas de Drosophila , ARN Helicasas , ARN Bicatenario , ARN Interferente Pequeño , Proteínas de Unión al ARN , Ribonucleasa III , Animales , Proteínas Argonautas/metabolismo , Emparejamiento Base , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/ultraestructura , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , MicroARNs/metabolismo , Multimerización de Proteína , ARN Helicasas/química , ARN Helicasas/metabolismo , ARN Helicasas/ultraestructura , Interferencia de ARN , ARN Bicatenario/química , ARN Bicatenario/metabolismo , ARN Bicatenario/ultraestructura , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/ultraestructura , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/ultraestructura , Complejo Silenciador Inducido por ARN/metabolismo , Ribonucleasa III/química , Ribonucleasa III/metabolismo , Ribonucleasa III/ultraestructuraRESUMEN
Around 250 million people are infected with hepatitis B virus (HBV) worldwide1, and 15 million may also carry the satellite virus hepatitis D virus (HDV), which confers even greater risk of severe liver disease2. The HBV receptor has been identified as sodium taurocholate co-transporting polypeptide (NTCP), which interacts directly with the first 48 amino acid residues of the N-myristoylated N-terminal preS1 domain of the viral large protein3. Despite the pressing need for therapeutic agents to counter HBV, the structure of NTCP remains unsolved. This 349-residue protein is closely related to human apical sodium-dependent bile acid transporter (ASBT), another member of the solute carrier family SLC10. Crystal structures have been reported of similar bile acid transporters from bacteria4,5, and these models are believed to resemble closely both NTCP and ASBT. Here we have used cryo-electron microscopy to solve the structure of NTCP bound to an antibody, clearly showing that the transporter has no equivalent of the first transmembrane helix found in other SLC10 proteins, and that the N terminus is exposed on the extracellular face. Comparison of our structure with those of related proteins indicates a common mechanism of bile acid transport, but the NTCP structure displays an additional pocket formed by residues that are known to interact with preS1, presenting new opportunities for structure-based drug design.
Asunto(s)
Ácidos y Sales Biliares , Microscopía por Crioelectrón , Virus de la Hepatitis B , Transportadores de Anión Orgánico Sodio-Dependiente , Receptores Virales , Simportadores , Anticuerpos , Ácidos y Sales Biliares/metabolismo , Virus de la Hepatitis B/metabolismo , Hepatocitos/metabolismo , Humanos , Transportadores de Anión Orgánico Sodio-Dependiente/química , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/ultraestructura , Receptores Virales/química , Receptores Virales/metabolismo , Receptores Virales/ultraestructura , Simportadores/química , Simportadores/metabolismo , Simportadores/ultraestructuraRESUMEN
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.
Asunto(s)
Microscopía por Crioelectrón , Activación del Canal Iónico , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Canales de Potasio Shal/química , Canales de Potasio Shal/metabolismo , Animales , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Femenino , Humanos , Proteínas de Interacción con los Canales Kv/química , Proteínas de Interacción con los Canales Kv/metabolismo , Modelos Moleculares , Complejos Multiproteicos/genética , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Oocitos/metabolismo , Canales de Potasio/química , Canales de Potasio/metabolismo , Unión Proteica , Canales de Potasio Shal/genética , Xenopus laevisRESUMEN
The mitochondrial outer membrane contains so-called ß-barrel proteins, which allow communication between the cytosol and the mitochondrial interior1-3. Insertion of ß-barrel proteins into the outer membrane is mediated by the multisubunit mitochondrial sorting and assembly machinery (SAM, also known as TOB)4-6. Here we use cryo-electron microscopy to determine the structures of two different forms of the yeast SAM complex at a resolution of 2.8-3.2 Å. The dimeric complex contains two copies of the ß-barrel channel protein Sam50-Sam50a and Sam50b-with partially open lateral gates. The peripheral membrane proteins Sam35 and Sam37 cap the Sam50 channels from the cytosolic side, and are crucial for the structural and functional integrity of the dimeric complex. In the second complex, Sam50b is replaced by the ß-barrel protein Mdm10. In cooperation with Sam50a, Sam37 recruits and traps Mdm10 by penetrating the interior of its laterally closed ß-barrel from the cytosolic side. The substrate-loaded SAM complex contains one each of Sam50, Sam35 and Sam37, but neither Mdm10 nor a second Sam50, suggesting that Mdm10 and Sam50b function as placeholders for a ß-barrel substrate released from Sam50a. Our proposed mechanism for dynamic switching of ß-barrel subunits and substrate explains how entire precursor proteins can fold in association with the mitochondrial machinery for ß-barrel assembly.
Asunto(s)
Microscopía por Crioelectrón , Mitocondrias/química , Mitocondrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Mitocondrias/ultraestructura , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/ultraestructura , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
Megalin (low-density lipoprotein receptor-related protein 2) is a giant glycoprotein of about 600 kDa, mediating the endocytosis of more than 60 ligands, including those of proteins, peptides, and drug compounds [S. Goto, M. Hosojima, H. Kabasawa, A. Saito, Int. J. Biochem. Cell Biol. 157, 106393 (2023)]. It is expressed predominantly in renal proximal tubule epithelial cells, as well as in the brain, lungs, eyes, inner ear, thyroid gland, and placenta. Megalin is also known to mediate the endocytosis of toxic compounds, particularly those that cause renal and hearing disorders [Y. Hori et al., J. Am. Soc. Nephrol. 28, 1783-1791 (2017)]. Genetic megalin deficiency causes Donnai-Barrow syndrome/facio-oculo-acoustico-renal syndrome in humans. However, it is not known how megalin interacts with such a wide variety of ligands and plays pathological roles in various organs. In this study, we elucidated the dimeric architecture of megalin, purified from rat kidneys, using cryoelectron microscopy. The maps revealed the densities of endogenous ligands bound to various regions throughout the dimer, elucidating the multiligand receptor nature of megalin. We also determined the structure of megalin in complex with receptor-associated protein, a molecular chaperone for megalin. The results will facilitate further studies on the pathophysiology of megalin-dependent multiligand endocytic pathways in multiple organs and will also be useful for the development of megalin-targeted drugs for renal and hearing disorders, Alzheimer's disease [B. V. Zlokovic et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4229-4234 (1996)], and other illnesses.
Asunto(s)
Microscopía por Crioelectrón , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Animales , Humanos , Ratas , Ligandos , Endocitosis , Agenesia del Cuerpo Calloso/metabolismo , Agenesia del Cuerpo Calloso/genética , Defectos Congénitos del Transporte Tubular Renal , Miopía , Hernias Diafragmáticas Congénitas , Proteinuria , Pérdida Auditiva SensorineuralRESUMEN
ATP11C is a member of the P4-ATPase flippase family that mediates translocation of phosphatidylserine (PtdSer) across the lipid bilayer. In order to characterize the structure and function of ATP11C in a model natural lipid environment, we revisited and optimized a quick procedure for reconstituting ATP11C into Nanodiscs using methyl-ß-cyclodextrin as a reagent for the detergent removal. ATP11C was efficiently reconstituted with the endogenous lipid, or the mixture of endogenous lipid and synthetic dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylserine (DOPS), all of which retained the ATPase activity. We obtained 3.4 Å and 3.9 Å structures using single-particle cryo-electron microscopy (cryo-EM) of AlF- and BeF-stabilized ATP11C transport intermediates, respectively, in a bilayer containing DOPS. We show that the latter exhibited a distended inner membrane around ATP11C transmembrane helix 2, possibly reflecting the perturbation needed for phospholipid release to the lipid bilayer. Our structures of ATP11C in the lipid membrane indicate that the membrane boundary varies upon conformational changes of the enzyme and is no longer flat around the protein, a change that likely contributes to phospholipid translocation across the membrane leaflets.
Asunto(s)
Adenosina Trifosfatasas , Membrana Dobles de Lípidos , Fosfolípidos , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismoRESUMEN
Channelrhodopsins (ChRs) are light-gated ion channels and central optogenetic tools that can control neuronal activity with high temporal resolution at the single-cell level. Although their application in optogenetics has rapidly progressed, it is unsolved how their channels open and close. ChRs transport ions through a series of interlocking elementary processes that occur over a broad time scale of subpicoseconds to seconds. During these processes, the retinal chromophore functions as a channel regulatory domain and transfers the optical input as local structural changes to the channel operating domain, the helices, leading to channel gating. Thus, the core question on channel gating dynamics is how the retinal chromophore structure changes throughout the photocycle and what rate-limits the kinetics. Here, we investigated the structural changes in the retinal chromophore of canonical ChR, C1C2, in all photointermediates using time-resolved resonance Raman spectroscopy. Moreover, to reveal the rate-limiting factors of the photocycle and channel gating, we measured the kinetic isotope effect of all photoreaction processes using laser flash photolysis and laser patch clamp, respectively. Spectroscopic and electrophysiological results provided the following understanding of the channel gating: the retinal chromophore highly twists upon the retinal Schiff base (RSB) deprotonation, causing the surrounding helices to move and open the channel. The ion-conducting pathway includes the RSB, where inflowing water mediates the proton to the deprotonated RSB. The twisting of the retinal chromophore relaxes upon the RSB reprotonation, which closes the channel. The RSB reprotonation rate-limits the channel closing.
Asunto(s)
Fenómenos Electrofisiológicos , Canales Iónicos , Channelrhodopsins/química , Protones , LuzRESUMEN
Lysophosphatidic acid (LPA) is a bioactive lipid composed of a phosphate group, a glycerol backbone, and a single acyl chain that varies in length and saturation. LPA activates six class A G-protein-coupled receptors to provoke various cellular reactions. Because LPA signalling has been implicated in cancer and fibrosis, the LPA receptors are regarded as promising drug targets. The six LPA receptors are subdivided into the endothelial differentiation gene (EDG) family (LPA1-LPA3) and the phylogenetically distant non-EDG family (LPA4-LPA6). The structure of LPA1 has enhanced our understanding of the EDG family of LPA receptors. By contrast, the functional and pharmacological characteristics of the non-EDG family of LPA receptors have remained unknown, owing to the lack of structural information. Although the non-EDG LPA receptors share sequence similarity with the P2Y family of nucleotide receptors, the LPA recognition mechanism cannot be deduced from the P2Y1 and P2Y12 structures because of the large differences in the chemical structures of their ligands. Here we determine the 3.2 Å crystal structure of LPA6, the gene deletion of which is responsible for congenital hair loss, to clarify the ligand recognition mechanism of the non-EDG family of LPA receptors. Notably, the ligand-binding pocket of LPA6 is laterally open towards the membrane, and the acyl chain of the lipid used for the crystallization is bound within this pocket, indicating the binding mode of the LPA acyl chain. Docking and mutagenesis analyses also indicated that the conserved positively charged residues within the central cavity recognize the phosphate head group of LPA by inducing an inward shift of transmembrane helices 6 and 7, suggesting that the receptor activation is triggered by this conformational rearrangement.
Asunto(s)
Lisofosfolípidos/química , Lisofosfolípidos/metabolismo , Receptores del Ácido Lisofosfatídico/química , Receptores del Ácido Lisofosfatídico/metabolismo , Alopecia/congénito , Alopecia/genética , Animales , Sitios de Unión , Membrana Celular/metabolismo , Cristalografía por Rayos X , Células HEK293 , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Mutagénesis , Filogenia , Estabilidad Proteica , Estructura Secundaria de Proteína , Receptores del Ácido Lisofosfatídico/genética , Especificidad por Sustrato , Pez Cebra/genéticaRESUMEN
Endothelin, a 21-amino-acid peptide, participates in various physiological processes, such as regulation of vascular tone, humoral homeostasis, neural crest cell development and neurotransmission. Endothelin and its G-protein-coupled receptor are involved in the development of various diseases, such as pulmonary arterial hypertension, and thus are important therapeutic targets. Here we report crystal structures of human endothelin type B receptor in the ligand-free form and in complex with the endogenous agonist endothelin-1. The structures and mutation analysis reveal the mechanism for the isopeptide selectivity between endothelin-1 and -3. Transmembrane helices 1, 2, 6 and 7 move and envelop the entire endothelin peptide, in a virtually irreversible manner. The agonist-induced conformational changes are propagated to the receptor core and the cytoplasmic G-protein coupling interface, and probably induce conformational flexibility in TM6. A comparison with the M2 muscarinic receptor suggests a shared mechanism for signal transduction in class A G-protein-coupled receptors.
Asunto(s)
Endotelina-1/metabolismo , Receptor de Endotelina B/química , Receptor de Endotelina B/metabolismo , Regulación Alostérica , Sitio Alostérico , Membrana Celular/metabolismo , Cristalografía por Rayos X , Endotelina-1/química , Endotelina-1/farmacología , Endotelina-3/química , Endotelina-3/metabolismo , Humanos , Ligandos , Modelos Moleculares , Conformación Proteica , Receptor de Endotelina B/agonistas , Receptor de Endotelina B/genética , Receptor Muscarínico M2/química , Receptor Muscarínico M2/metabolismo , Transducción de Señal , Especificidad por SustratoRESUMEN
The drug/metabolite transporter (DMT) superfamily is a large group of membrane transporters ubiquitously found in eukaryotes, bacteria and archaea, and includes exporters for a remarkably wide range of substrates, such as toxic compounds and metabolites. YddG is a bacterial DMT protein that expels aromatic amino acids and exogenous toxic compounds, thereby contributing to cellular homeostasis. Here we present structural and functional analyses of YddG. Using liposome-based analyses, we show that Escherichia coli and Starkeya novella YddG export various amino acids. The crystal structure of S. novella YddG at 2.4 Å resolution reveals a new membrane transporter topology, with ten transmembrane segments in an outward-facing state. The overall structure is basket-shaped, with a large substrate-binding cavity at the centre of the molecule, and is composed of inverted structural repeats related by two-fold pseudo-symmetry. On the basis of this intramolecular symmetry, we propose a structural model for the inward-facing state and a mechanism of the conformational change for substrate transport, which we confirmed by biochemical analyses. These findings provide a structural basis for the mechanism of transport of DMT superfamily proteins.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Alphaproteobacteria/química , Alphaproteobacteria/metabolismo , Transporte Biológico , Cristalografía por Rayos X , Escherichia coli/química , Escherichia coli/metabolismo , Liposomas/química , Liposomas/metabolismo , Modelos Moleculares , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.
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Flavobacteriaceae/química , Bombas Iónicas/química , Bombas Iónicas/efectos de la radiación , Luz , Rodopsina/química , Rodopsina/efectos de la radiación , Sodio/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Bombas Iónicas/genética , Bombas Iónicas/metabolismo , Transporte Iónico/genética , Transporte Iónico/efectos de la radiación , Modelos Biológicos , Modelos Moleculares , Mutagénesis/genética , Optogenética , Potasio/metabolismo , Conformación Proteica , Ingeniería de Proteínas , Retinaldehído/química , Retinaldehído/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Bases de Schiff , Relación Estructura-ActividadRESUMEN
Novel pyrrole derivatives were discovered as potent agonists of the niacin receptor, GPR109A. During the derivatization, compound 16 was found to be effective both in vitro and in vivo. The compound 16 exhibited a significant reduction of the non-esterified fatty acid in human GPR109A transgenic rats, and the duration of its in vivo efficacy was much longer than niacin.
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Agonistas Nicotínicos/química , Pirroles/química , Receptores Acoplados a Proteínas G/agonistas , Animales , Diseño de Fármacos , Ácidos Grasos no Esterificados/metabolismo , Humanos , Lipólisis/efectos de los fármacos , Agonistas Nicotínicos/metabolismo , Agonistas Nicotínicos/farmacología , Pirroles/metabolismo , Pirroles/farmacología , Ratas , Ratas Transgénicas , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-ActividadRESUMEN
Since it was recently reported that an antibody for proprotein convertase subtilisin/kexin type 9 (PCSK9) reduces the risk of cardiovascular events in a clinical context, PCSK9 inhibition is thought to be an attractive therapy for dyslipidemia. In the present study, we created a novel small biologic alternative to PCSK9 antibodies called DS-9001a, comprising an albumin binding domain fused to an artificial lipocalin mutein (ABD-fused Anticalin protein), which can be produced by a microbial production system. DS-9001a strongly interfered with PCSK9 binding to low-density-lipoprotein receptor (LDL-R) and PCSK9-mediated degradation of LDL-R. In cynomolgus monkeys, single DS-9001a administration significantly reduced the serum LDL-C level up to 21 days (62.4% reduction at the maximum). Moreover, DS-9001a reduced plasma non-high-density-lipoprotein cholesterol and oxidized LDL levels, and their further reductions were observed when atorvastatin and DS-9001a were administered in combination in human cholesteryl ester transfer protein/ApoB double transgenic mice. Additionally, their reductions on the combination of atorvastatin and DS-9001a were more pronounced than those on the combination of atorvastatin and anacetrapib. Besides its favorable pharmacologic profile, DS-9001a has a lower molecular weight (about 22 kDa), yielding a high stoichiometric drug concentration that might result in a smaller administration volume than that in existing antibody therapy. Since bacterial production systems are viewed as more suited to mass production at low cost, DS-9001a may provide a new therapeutic option to treat patients with dyslipidemia. In addition, considering the growing demand for antibody-like drugs, ABD-fused Anticalin proteins could represent a promising new class of small biologic molecules.