RESUMEN
Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Microscopía por Crioelectrón , Prolina , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/genética , Prolina/metabolismo , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , COVID-19/virología , COVID-19/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/química , Modelos MolecularesRESUMEN
Two pore channels are lysosomal cation channels with crucial roles in tumor angiogenesis and viral release from endosomes. Inhibition of the two-pore channel 2 (TPC2) has emerged as potential therapeutic strategy for the treatment of cancers and viral infections, including Ebola and COVID-19. Here, we demonstrate that antagonist SG-094, a synthetic analog of the Chinese alkaloid medicine tetrandrine with increased potency and reduced toxicity, induces asymmetrical structural changes leading to a single binding pocket at only one intersubunit interface within the asymmetrical dimer. Supported by functional characterization of mutants by Ca2+ imaging and patch clamp experiments, we identify key residues in S1 and S4 involved in compound binding to the voltage sensing domain II. SG-094 arrests IIS4 in a downward shifted state which prevents pore opening via the IIS4/S5 linker, hence resembling gating modifiers of canonical VGICs. These findings may guide the rational development of new therapeutics antagonizing TPC2 activity.
Asunto(s)
Canales de Calcio , Humanos , Canales de Calcio/metabolismo , Canales de Calcio/química , Sitios de Unión , Lisosomas/metabolismo , Células HEK293 , Unión Proteica , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/metabolismo , Modelos Moleculares , Canales de Dos PorosRESUMEN
Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K+ channel superfamily. To address the issue of selectivity, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.
Asunto(s)
Canales de Potasio de Dominio Poro en Tándem , Anticuerpos de Dominio Único , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Humanos , Cristalografía por Rayos X , Animales , Microscopía por Crioelectrón , Células HEK293 , Modelos MolecularesRESUMEN
In this study, we present the structures of human urea transporters UT-A and UT-B to characterize them at molecular level and to detail the mechanism of UT-B inhibition by its selective inhibitor, UTBinh-14. High-resolution structures of both transporters establish the structural basis for the inhibitor's selectivity to UT-B, and the identification of multiple binding sites for the inhibitor will aid with the development of drug lead molecules targeting both transporters. Our study also discovers phospholipids associating with the urea transporters by combining structural observations, native MS, and lipidomics analysis. These insights improve our understanding of urea transporter function at a molecular level and provide a blueprint for a structure-guided design of therapeutics targeting these transporters.
Asunto(s)
Proteínas de Transporte de Membrana , Urea , Humanos , Proteínas de Transporte de Membrana/metabolismo , Sitios de Unión , Urea/farmacología , Urea/metabolismo , Transportadores de UreaRESUMEN
Very long chain fatty acids (VLCFAs) are essential building blocks for the synthesis of ceramides and sphingolipids. The first step in the fatty acid elongation cycle is catalyzed by the 3-keto acyl-coenzyme A (CoA) synthases (in mammals, ELOVL elongases). Although ELOVLs are implicated in common diseases, including insulin resistance, hepatic steatosis and Parkinson's, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which comprises an inverted transmembrane barrel surrounding a 35-Å long tunnel containing a covalently attached product analogue. The structure reveals the substrate-binding sites in the narrow tunnel and an active site deep in the membrane. We demonstrate that chain elongation proceeds via an acyl-enzyme intermediate involving the second histidine in the canonical HxxHH motif. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate, such as X-linked adrenoleukodystrophy.
Asunto(s)
Elongasas de Ácidos Grasos/química , Ácidos Grasos/metabolismo , Adrenoleucodistrofia/enzimología , Animales , Sitios de Unión , Dominio Catalítico , Clonación Molecular , Coenzima A/metabolismo , Cristalografía por Rayos X , Elongasas de Ácidos Grasos/antagonistas & inhibidores , Elongasas de Ácidos Grasos/metabolismo , Células HEK293 , Histidina/química , Humanos , Imidazoles/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Células Sf9 , Espectrometría de Masa por Ionización de Electrospray/métodos , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
TWIK-related acid-sensitive potassium (TASK) channels-members of the two pore domain potassium (K2P) channel family-are found in neurons1, cardiomyocytes2-4 and vascular smooth muscle cells5, where they are involved in the regulation of heart rate6, pulmonary artery tone5,7, sleep/wake cycles8 and responses to volatile anaesthetics8-11. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli12-15. Unlike other K2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation16. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate-which we designate as an 'X-gate'-created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (243VLRFMT248) that are essential for responses to volatile anaesthetics10, neurotransmitters13 and G-protein-coupled receptors13. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders.
Asunto(s)
Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/química , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/química , Anestésicos/farmacología , Animales , Cristalografía por Rayos X , Conductividad Eléctrica , Femenino , Humanos , Activación del Canal Iónico/efectos de los fármacos , Modelos Moleculares , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Xenopus laevisRESUMEN
Polycystin-2 (PC2) is a transient receptor potential (TRP) channel present in ciliary membranes of the kidney. PC2 shares a transmembrane fold with other TRP channels, in addition to an extracellular domain found in TRPP and TRPML channels. Using molecular dynamics (MD) simulations and cryoelectron microscopy we identify and characterize PIP2 and cholesterol interactions with PC2. PC2 is revealed to have a PIP binding site close to the equivalent vanilloid/lipid binding site in the TRPV1 channel. A 3.0-Å structure reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are characterized by MD simulations. The two classes of lipid binding sites are compared with sites observed in other TRPs and in Kv channels. These findings suggest PC2, in common with other ion channels, may be modulated by both PIPs and cholesterol, and position PC2 within an emerging model of the roles of lipids in the regulation and organization of ciliary membranes.
Asunto(s)
Colesterol/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Canales Catiónicos TRPP/química , Canales Catiónicos TRPP/metabolismo , Animales , Sitios de Unión , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Estructura Secundaria de Proteína , Células Sf9RESUMEN
Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity.
Asunto(s)
Anoctaminas/metabolismo , Retículo Endoplásmico/metabolismo , Lípidos/química , Proteínas de Transferencia de Fosfolípidos/metabolismo , Secuencia de Aminoácidos , Animales , Anoctaminas/química , Anoctaminas/genética , Células COS , Calcio/química , Línea Celular Tumoral , Chlorocebus aethiops , Cristalografía por Rayos X , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/genética , Homología de Secuencia de Aminoácido , Células Sf9 , SpodopteraRESUMEN
Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go-related gene) channels and calcium (Ca2+)-activated big-conductance potassium (BK)-type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.
Asunto(s)
Clorobencenos/farmacología , Canal de Potasio ERG1/agonistas , Canal de Potasio ERG1/química , Activación del Canal Iónico/efectos de los fármacos , Canales de Potasio de Gran Conductancia Activados por el Calcio/agonistas , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Tetrahidronaftalenos/farmacología , Tetrazoles/farmacología , Tiourea/análogos & derivados , ortoaminobenzoatos/farmacología , Animales , Células CHO , Clorobencenos/química , Cricetulus , Cristalografía por Rayos X , Diseño de Fármacos , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Dominios Proteicos , Tetrahidronaftalenos/química , Tetrazoles/química , Tiourea/química , Tiourea/farmacología , Xenopus , ortoaminobenzoatos/químicaRESUMEN
Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Trastornos Congénitos de Glicosilación/metabolismo , Inhibidores Enzimáticos/farmacología , N-Acetilglucosaminiltransferasas/química , Animales , Antibióticos Antituberculosos/química , Sitios de Unión , Trastornos Congénitos de Glicosilación/genética , Inhibidores Enzimáticos/química , Femenino , Células HEK293 , Células Hep G2 , Humanos , Metabolismo de los Lípidos , Ratones , Simulación del Acoplamiento Molecular , Mutación , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Unión Proteica , Células Sf9 , Spodoptera , Tunicamicina/química , Tunicamicina/farmacología , Uridina Difosfato Ácido Glucurónico/química , Uridina Difosfato Ácido Glucurónico/metabolismoRESUMEN
The mechanosensitive two-pore domain (K2P) K+ channels (TREK-1, TREK-2, and TRAAK) are important for mechanical and thermal nociception. However, the mechanisms underlying their gating by membrane stretch remain controversial. Here we use molecular dynamics simulations to examine their behavior in a lipid bilayer. We show that TREK-2 moves from the "down" to "up" conformation in direct response to membrane stretch, and examine the role of the transmembrane pressure profile in this process. Furthermore, we show how state-dependent interactions with lipids affect the movement of TREK-2, and how stretch influences both the inner pore and selectivity filter. Finally, we present functional studies that demonstrate why direct pore block by lipid tails does not represent the principal mechanism of mechanogating. Overall, this study provides a dynamic structural insight into K2P channel mechanosensitivity and illustrates how the structure of a eukaryotic mechanosensitive ion channel responds to changes in forces within the bilayer.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio de Dominio Poro en Tándem/química , Humanos , Membrana Dobles de Lípidos/química , Mecanotransducción Celular , Canales de Potasio de Dominio Poro en Tándem/metabolismoRESUMEN
Polycystin-2 (PC2), a calcium-activated cation TRP channel, is involved in diverse Ca2+ signaling pathways. Malfunctioning Ca2+ regulation in PC2 causes autosomal-dominant polycystic kidney disease. Here we report two cryo-EM structures of distinct channel states of full-length human PC2 in complex with lipids and cations. The structures reveal conformational differences in the selectivity filter and in the large exoplasmic domain (TOP domain), which displays differing N-glycosylation. The more open structure has one cation bound below the selectivity filter (single-ion mode, PC2SI), whereas multiple cations are bound along the translocation pathway in the second structure (multi-ion mode, PC2MI). Ca2+ binding at the entrance of the selectivity filter suggests Ca2+ blockage in PC2MI, and we observed density for the Ca2+-sensing C-terminal EF hand in the unblocked PC2SI state. The states show altered interactions of lipids with the pore loop and TOP domain, thus reflecting the functional diversity of PC2 at different locations, owing to different membrane compositions.
Asunto(s)
Canales Catiónicos TRPP/química , Sitios de Unión , Calcio/química , Señalización del Calcio , Microscopía por Crioelectrón , Glicosilación , Células HEK293 , Humanos , Modelos Moleculares , Ácidos Fosfatidicos/química , Fosfatidilcolinas/química , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Estructura Cuaternaria de ProteínaRESUMEN
Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli.
Asunto(s)
Canales Catiónicos TRPP/química , Animales , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Mutación Missense , Riñón Poliquístico Autosómico Dominante/genética , Conformación Proteica en Hélice alfa , Dominios Proteicos , Células Sf9 , Spodoptera , Canales Catiónicos TRPP/genéticaRESUMEN
Heavy-atom derivatization is one of the oldest techniques for obtaining phase information for protein crystals and, although it is no longer the first choice, it remains a useful technique for obtaining phases for unknown structures and for low-resolution data sets. It is also valuable for confirming the chain trace in low-resolution electron-density maps. This overview provides a summary of the technique and is aimed at first-time users of the method. It includes guidelines on when to use it, which heavy atoms are most likely to work, how to prepare heavy-atom solutions, how to derivatize crystals and how to determine whether a crystal is in fact a derivative.
Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas/química , Animales , Bromo/química , Humanos , Yodo/química , Metales Pesados/química , Metionina/química , Gases Nobles/química , Conformación Proteica , Selenometionina/químicaRESUMEN
RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration.
Asunto(s)
ADN Helicasas/química , ADN/química , RecQ Helicasas/química , Animales , Cromatografía en Gel , Cristalización , Cristalografía por Rayos X , ADN Cruciforme/fisiología , ADN de Cadena Simple/química , Escherichia coli/metabolismo , Humanos , Insectos , Conformación Molecular , Desnaturalización de Ácido Nucleico , Nucleótidos/química , Unión Proteica , Estructura Terciaria de Proteína , Zinc/químicaRESUMEN
TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio de Dominio Poro en Tándem/química , Secuencia de Aminoácidos , Ácido Araquidónico/farmacología , Sitios de Unión , Cristalografía por Rayos X , Fluoxetina/análogos & derivados , Fluoxetina/química , Fluoxetina/metabolismo , Fluoxetina/farmacología , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Potasio/metabolismo , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Modern synchrotron beamlines offer instrumentation of unprecedented quality, which in turn encourages increasingly marginal experiments, and for these, as much as ever, the ultimate success of data collection depends on the experience, but especially the care, of the experimenter. A representative set of difficult cases has been encountered at the Structural Genomics Consortium, a worldwide structural genomics initiative of which the Oxford site currently deposits three novel human structures per month. Achieving this target relies heavily on frequent visits to the Diamond Light Source, and the variety of crystal systems still demand customized data collection, diligent checks and careful planning of each experiment. Here, an overview is presented of the techniques and procedures that have been refined over the years and that are considered synchrotron best practice.
Asunto(s)
Cristalografía por Rayos X/instrumentación , Cristalografía por Rayos X/métodos , Recolección de Datos/métodos , Procesamiento Automatizado de Datos/métodos , Sustancias Macromoleculares/química , Sincrotrones/instrumentación , Biología Computacional , Genómica , HumanosRESUMEN
ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Conformación Molecular , Nucleótidos/química , Estructura Terciaria de Proteína , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Cristalografía por Rayos X , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutación , Nucleótidos/metabolismo , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Unión Proteica , Homología de Secuencia de Aminoácido , Células Sf9RESUMEN
Mutations in the nuclear membrane zinc metalloprotease ZMPSTE24 lead to diseases of lamin processing (laminopathies), such as the premature aging disease progeria and metabolic disorders. ZMPSTE24 processes prelamin A, a component of the nuclear lamina intermediate filaments, by cleaving it at two sites. Failure of this processing results in accumulation of farnesylated, membrane-associated prelamin A. The 3.4 angstrom crystal structure of human ZMPSTE24 has a seven transmembrane α-helical barrel structure, surrounding a large, water-filled, intramembrane chamber, capped by a zinc metalloprotease domain with the catalytic site facing into the chamber. The 3.8 angstrom structure of a complex with a CSIM tetrapeptide showed that the mode of binding of the substrate resembles that of an insect metalloprotease inhibitor in thermolysin. Laminopathy-associated mutations predicted to reduce ZMPSTE24 activity map to the zinc metalloprotease peptide-binding site and to the bottom of the chamber.
Asunto(s)
Proteínas de la Membrana/química , Errores Innatos del Metabolismo/metabolismo , Metaloendopeptidasas/química , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Lamina Tipo A , Proteínas de la Membrana/genética , Errores Innatos del Metabolismo/genética , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Progeria/genética , Progeria/metabolismo , Conformación Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Especificidad por Sustrato , Termolisina/químicaRESUMEN
Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein "3-box" motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.