RESUMEN
Predicting protein function is crucial for understanding biological life processes, preventing diseases and developing new drug targets. In recent years, methods based on sequence, structure and biological networks for protein function annotation have been extensively researched. Although obtaining a protein in three-dimensional structure through experimental or computational methods enhances the accuracy of function prediction, the sheer volume of proteins sequenced by high-throughput technologies presents a significant challenge. To address this issue, we introduce a deep neural network model DeepSS2GO (Secondary Structure to Gene Ontology). It is a predictor incorporating secondary structure features along with primary sequence and homology information. The algorithm expertly combines the speed of sequence-based information with the accuracy of structure-based features while streamlining the redundant data in primary sequences and bypassing the time-consuming challenges of tertiary structure analysis. The results show that the prediction performance surpasses state-of-the-art algorithms. It has the ability to predict key functions by effectively utilizing secondary structure information, rather than broadly predicting general Gene Ontology terms. Additionally, DeepSS2GO predicts five times faster than advanced algorithms, making it highly applicable to massive sequencing data. The source code and trained models are available at https://github.com/orca233/DeepSS2GO.
Asunto(s)
Algoritmos , Biología Computacional , Redes Neurales de la Computación , Estructura Secundaria de Proteína , Proteínas , Proteínas/química , Proteínas/metabolismo , Proteínas/genética , Biología Computacional/métodos , Bases de Datos de Proteínas , Ontología de Genes , Análisis de Secuencia de Proteína/métodos , Programas InformáticosRESUMEN
High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC) function and suppress inflammation, but their utility in treating EC dysfunction has not been fully explored. Here, we describe a fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1 (ApoA1), the principal structural protein of HDL that forms lipid nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine 1-phosphate (S1P). A1M forms HDL-like particles, binds to S1P, and is signaling competent. Molecular dynamics simulations showed that the S1P-bound ApoM moiety in A1M efficiently activated EC surface receptors. Treatment of human umbilical vein ECs with A1M-S1P stimulated barrier function either alone or cooperatively with other barrier-enhancing molecules, including the stable prostacyclin analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P injection into mice during sterile inflammation suppressed neutrophil influx and inflammatory mediator secretion. Moreover, systemic A1M administration led to a sustained increase in circulating HDL-bound S1P and suppressed inflammation in a murine model of LPS-induced endotoxemia. We propose that A1M administration may enhance vascular endothelial barrier function, suppress cytokine storm, and promote resilience of the vascular endothelium.
Asunto(s)
Apolipoproteínas , Lipocalinas , Humanos , Ratones , Animales , Apolipoproteínas/metabolismo , Apolipoproteínas/farmacología , Lipocalinas/metabolismo , Lipocalinas/farmacología , Receptores de Lisoesfingolípidos/metabolismo , Apolipoproteínas M , Inflamación , Lipoproteínas HDL/farmacología , Lipoproteínas HDL/metabolismo , Lisofosfolípidos/farmacología , Lisofosfolípidos/metabolismo , EsfingosinaRESUMEN
Mitochondrial adenosine triphosphate (ATP) synthase uses the proton gradient across the inner mitochondrial membrane to synthesize ATP. Structural and single molecule studies conducted mostly at neutral or basic pH have provided details of the reaction mechanism of ATP synthesis. However, pH of the mitochondrial matrix is slightly acidic during hypoxia and pH-dependent conformational changes in the ATP synthase have been reported. Here we use single-particle cryo-EM to analyze the conformational ensemble of the yeast (Saccharomyces cerevisiae) ATP synthase at pH 6. Of the four conformations resolved in this study, three are reaction intermediates. In addition to canonical catalytic dwell and binding dwell structures, we identify two unique conformations with nearly identical positions of the central rotor but different catalytic site conformations. These structures provide new insights into the catalytic mechanism of the ATP synthase and highlight elastic coupling between the catalytic and proton translocating domains.
Asunto(s)
Protones , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/metabolismo , ATPasas de Translocación de Protón Mitocondriales/química , Conformación Proteica , Concentración de Iones de HidrógenoRESUMEN
In response to cold, mammals activate brown fat for respiratory-dependent thermogenesis reliant on the electron transport chain (1, 2). Yet, the structural basis of respiratory complex adaptation to cold remains elusive. Herein we combined thermoregulatory physiology and cryo-EM to study endogenous respiratory supercomplexes exposed to different temperatures. A cold-induced conformation of CI:III 2 (termed type 2) was identified with a â¼25° rotation of CIII 2 around its inter-dimer axis, shortening inter-complex Q exchange space, and exhibiting different catalytic states which favor electron transfer. Large-scale supercomplex simulations in lipid membrane reveal how unique lipid-protein arrangements stabilize type 2 complexes to enhance catalytic activity. Together, our cryo-EM studies, multiscale simulations and biochemical analyses unveil the mechanisms and dynamics of respiratory adaptation at the structural and energetic level.
RESUMEN
As the first identified multidrug efflux pump in Mycobacterium tuberculosis (Mtb), EfpA is an essential protein and promising drug target. However, the functional and inhibitory mechanisms of EfpA are poorly understood. Herein we report cryo-EM structures of EfpA in outward-open conformation, either bound to three endogenous lipids or the inhibitor BRD-8000.3. Three lipids inside EfpA span from the inner leaflet to the outer leaflet of the membrane. BRD-8000.3 occupies one lipid site at the level of inner membrane leaflet, competitively inhibiting lipid binding. EfpA resembles the related lysophospholipid transporter MFSD2A in both overall structure and lipid binding sites, and may function as a lipid flippase. Combining AlphaFold-predicted EfpA structure, which is inward-open, we propose a complete conformational transition cycle for EfpA. Together, our results provide a structural and mechanistic foundation to comprehend EfpA function and develop EfpA-targeting anti-TB drugs.
RESUMEN
Cells remodel glycerophospholipid acyl chains via the Lands cycle to adjust membrane properties. Membrane-bound O-acyltransferase (MBOAT) 7 acylates lyso-phosphatidylinositol (lyso-PI) with arachidonyl-CoA. MBOAT7 mutations cause brain developmental disorders, and reduced expression is linked to fatty liver disease. In contrast, increased MBOAT7 expression is linked to hepatocellular and renal cancers. The mechanistic basis of MBOAT7 catalysis and substrate selectivity are unknown. Here, we report the structure and a model for the catalytic mechanism of human MBOAT7. Arachidonyl-CoA and lyso-PI access the catalytic center through a twisted tunnel from the cytosol and lumenal sides, respectively. N-terminal residues on the ER lumenal side determine phospholipid headgroup selectivity: swapping them between MBOATs 1, 5, and 7 converts enzyme specificity for different lyso-phospholipids. Finally, the MBOAT7 structure and virtual screening enabled identification of small-molecule inhibitors that may serve as lead compounds for pharmacologic development.
Asunto(s)
Encefalopatías , Neoplasias Renales , Humanos , Fosfatidilinositoles , Glicerofosfolípidos , Fosfolípidos , Catálisis , Aciltransferasas/genética , Proteínas de la Membrana/genéticaRESUMEN
Inhibitors of triacylglycerol (TG) synthesis have been developed to treat metabolism-related diseases, but we know little about their mechanisms of action. Here, we report cryo-EM structures of the TG-synthesis enzyme acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a membrane bound O-acyltransferase (MBOAT), in complex with two different inhibitors, T863 and DGAT1IN1. Each inhibitor binds DGAT1's fatty acyl-CoA substrate binding tunnel that opens to the cytoplasmic side of the ER. T863 blocks access to the tunnel entrance, whereas DGAT1IN1 extends further into the enzyme, with an amide group interacting with more deeply buried catalytic residues. A survey of DGAT1 inhibitors revealed that this amide group may serve as a common pharmacophore for inhibition of MBOATs. The inhibitors were minimally active against the related MBOAT acyl-CoA:cholesterol acyltransferase 1 (ACAT1), yet a single-residue mutation sensitized ACAT1 for inhibition. Collectively, our studies provide a structural foundation for developing DGAT1 and other MBOAT inhibitors.
Asunto(s)
Aciltransferasas , Diacilglicerol O-Acetiltransferasa , Aciltransferasas/metabolismo , Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Lipogénesis , Esterol O-Aciltransferasa/química , TriglicéridosRESUMEN
The human ATP-binding cassette (ABC) transporter ABCA1 plays a critical role in lipid homeostasis as it extracts sterols and phospholipids from the plasma membrane for excretion to the extracellular apolipoprotein A-I and subsequent formation of high-density lipoprotein (HDL) particles. Deleterious mutations of ABCA1 lead to sterol accumulation and are associated with atherosclerosis, poor cardiovascular outcomes, cancer, and Alzheimer's disease. The mechanism by which ABCA1 drives lipid movement is poorly understood, and a unified platform to produce active ABCA1 protein for both functional and structural studies has been missing. In this work, we established a stable expression system for both a human cell-based sterol export assay and protein purification for in vitro biochemical and structural studies. ABCA1 produced in this system was active in sterol export and displayed enhanced ATPase activity after reconstitution into a lipid bilayer. Our single-particle cryo-EM study of ABCA1 in nanodiscs showed protein induced membrane curvature, revealed multiple distinct conformations, and generated a structure of nanodisc-embedded ABCA1 at 4.0-Å resolution representing a previously unknown conformation. Comparison of different ABCA1 structures and molecular dynamics simulations demonstrates both concerted domain movements and conformational variations within each domain. Taken together, our platform for producing and characterizing ABCA1 in a lipid membrane enabled us to gain important mechanistic and structural insights and paves the way for investigating modulators that target the functions of ABCA1.
Asunto(s)
Transportador 1 de Casete de Unión a ATP , Membrana Celular , Lípidos de la Membrana , Imagen Individual de Molécula , Esteroles , Humanos , Apolipoproteína A-I/metabolismo , Transportador 1 de Casete de Unión a ATP/química , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Membrana Celular/química , Fosfolípidos/química , Esteroles/química , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Mutación , Membrana Dobles de Lípidos/química , Imagen Individual de Molécula/métodosRESUMEN
MsbA is a member of the ATP-binding cassette (ABC) transporter family and harnesses the energy from adenosine triphosphate (ATP) binding and hydrolysis to flip lipopolysaccharide (LPS) across the cytoplasmic membrane in Gram-negative bacteria. MsbA is an essential component of the bacterial envelope biogenesis pathway and an attractive target for developing novel antibiotics against multidrug-resistant strains. Structural characterization of MsbA in different conformations provides crucial insights in understanding druggable pockets and mechanisms of inhibition of this transporter. Recent advances in membrane-mimetic environments and cryo-EM data acquisition and processing have enabled high-resolution imaging of MsbA in complex with its native LPS substrate. Despite these technical advances, MsbA remains a challenging target for cryo-EM analysis due to its small size and extraordinary conformational flexibility. Herein, we provide a protocol for the purification and incorporation of MsbA in lipid nanodiscs, cryo-EM sample preparation, and cryo-EM image processing. The method outlined here is generalizable to the study of other bacterial ABC transporters, including the LPS extractor LptB2FGC.
Asunto(s)
Proteínas de Escherichia coli , Lipopolisacáridos , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipopolisacáridos/metabolismoRESUMEN
High-resolution structural studies are essential for understanding the folding and function of diverse RNAs. Herein, we present a nanoarchitectural engineering strategy for efficient structural determination of RNA-only structures using single-particle cryogenic electron microscopy (cryo-EM). This strategy-ROCK (RNA oligomerization-enabled cryo-EM via installing kissing loops)-involves installing kissing-loop sequences onto the functionally nonessential stems of RNAs for homomeric self-assembly into closed rings with multiplied molecular weights and mitigated structural flexibility. ROCK enables cryo-EM reconstruction of the Tetrahymena group I intron at 2.98-Å resolution overall (2.85 Å for the core), allowing de novo model building of the complete RNA, including the previously unknown peripheral domains. ROCK is further applied to two smaller RNAs-the Azoarcus group I intron and the FMN riboswitch, revealing the conformational change of the former and the bound ligand in the latter. ROCK holds promise to greatly facilitate the use of cryo-EM in RNA structural studies.
Asunto(s)
ARN , Riboswitch , Microscopía por Crioelectrón , Ligandos , ARN/genética , Imagen Individual de MoléculaRESUMEN
Lipid droplets (LDs) form in the endoplasmic reticulum by phase separation of neutral lipids. This process is facilitated by the seipin protein complex, which consists of a ring of seipin monomers, with a yet unclear function. Here, we report a structure of S. cerevisiae seipin based on cryogenic-electron microscopy and structural modeling data. Seipin forms a decameric, cage-like structure with the lumenal domains forming a stable ring at the cage floor and transmembrane segments forming the cage sides and top. The transmembrane segments interact with adjacent monomers in two distinct, alternating conformations. These conformations result from changes in switch regions, located between the lumenal domains and the transmembrane segments, that are required for seipin function. Our data indicate a model for LD formation in which a closed seipin cage enables triacylglycerol phase separation and subsequently switches to an open conformation to allow LD growth and budding.
Asunto(s)
Subunidades gamma de la Proteína de Unión al GTP , Gotas Lipídicas , Retículo Endoplásmico/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/química , Gotas Lipídicas/química , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos , Proteínas de la Membrana/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
CACNA1I is implicated in the susceptibility to schizophrenia by large-scale genetic association studies of single nucleotide polymorphisms. However, the channelopathy of CACNA1I in schizophrenia is unknown. CACNA1I encodes CaV3.3, a neuronal voltage-gated calcium channel that underlies a subtype of T-type current that is important for neuronal excitability in the thalamic reticular nucleus and other regions of the brain. Here, we present an extensive functional characterization of 57 naturally occurring rare and common missense variants of CACNA1I derived from a Swedish schizophrenia cohort of more than 10 000 individuals. Our analysis of this allelic series of coding CACNA1I variants revealed that reduced CaV3.3 channel current density was the dominant phenotype associated with rare CACNA1I coding alleles derived from control subjects, whereas rare CACNA1I alleles from schizophrenia patients encoded CaV3.3 channels with altered responses to voltages. CACNA1I variants associated with altered current density primarily impact the ionic channel pore and those associated with altered responses to voltage impact the voltage-sensing domain. CaV3.3 variants associated with altered voltage dependence of the CaV3.3 channel and those associated with peak current density deficits were significantly segregated across affected and unaffected groups (Fisher's exact test, P = 0.034). Our results, together with recent data from the SCHEMA (Schizophrenia Exome Sequencing Meta-Analysis) cohort, suggest that reduced CaV3.3 function may protect against schizophrenia risk in rare cases. We subsequently modelled the effect of the biophysical properties of CaV3.3 channel variants on thalamic reticular nucleus excitability and found that compared with common variants, ultrarare CaV3.3-coding variants derived from control subjects significantly decreased thalamic reticular nucleus excitability (P = 0.011). When all rare variants were analysed, there was a non-significant trend between variants that reduced thalamic reticular nucleus excitability and variants that either had no effect or increased thalamic reticular nucleus excitability across disease status. Taken together, the results of our functional analysis of an allelic series of >50 CACNA1I variants in a schizophrenia cohort reveal that loss of function of CaV3.3 is a molecular phenotype associated with reduced disease risk burden, and our approach may serve as a template strategy for channelopathies in polygenic disorders.
Asunto(s)
Canales de Calcio Tipo T , Canalopatías , Esquizofrenia , Alelos , Canales de Calcio Tipo T/genética , Canalopatías/genética , Humanos , Mutación Missense , Esquizofrenia/genética , SueciaRESUMEN
ATP-binding cassette (ABC) transporters couple adenosine 5'-triphosphate (ATP) hydrolysis to substrate transport across biological membranes. Although many are promising drug targets, their mechanisms of modulation by small-molecule inhibitors remain largely unknown. Two first-generation inhibitors of the MsbA transporter, tetrahydrobenzothiophene 1 (TBT1) and G247, induce opposite effects on ATP hydrolysis. Using single-particle cryoelectron microscopy and functional assays, we show that TBT1 and G247 bind adjacent yet separate pockets in the MsbA transmembrane domains. Two TBT1 molecules asymmetrically occupy the substrate-binding site, which leads to a collapsed inward-facing conformation with decreased distance between the nucleotide-binding domains (NBDs). By contrast, two G247 molecules symmetrically increase NBD distance in a wide inward-open state of MsbA. The divergent mechanisms of action of these MsbA inhibitors provide important insights into ABC transporter pharmacology.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfatasas/metabolismo , Regulación Alostérica , Proteínas Bacterianas/química , Sitios de Unión , Microscopía por Crioelectrón , Descubrimiento de Drogas , Imagenología Tridimensional , Lipopolisacáridos/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Bibliotecas de Moléculas PequeñasRESUMEN
Lipoproteins are important for bacterial growth and antibiotic resistance. These proteins use lipid acyl chains attached to the N-terminal cysteine residue to anchor on the outer surface of cytoplasmic membrane. In Gram-negative bacteria, many lipoproteins are transported to the outer membrane (OM), a process dependent on the ATP-binding cassette (ABC) transporter LolCDE which extracts the OM-targeted lipoproteins from the cytoplasmic membrane. Lipid-anchored proteins pose a unique challenge for transport machinery as they have both hydrophobic lipid moieties and soluble protein component, and the underlying mechanism is poorly understood. Here we determined the cryo-EM structures of nanodisc-embedded LolCDE in the nucleotide-free and nucleotide-bound states at 3.8-Å and 3.5-Å resolution, respectively. The structural analyses, together with biochemical and mutagenesis studies, uncover how LolCDE recognizes its substrate by interacting with the lipid and N-terminal peptide moieties of the lipoprotein, and identify the amide-linked acyl chain as the key element for LolCDE interaction. Upon nucleotide binding, the transmembrane helices and the periplasmic domains of LolCDE undergo large-scale, asymmetric movements, resulting in extrusion of the captured lipoprotein. Comparison of LolCDE and MacB reveals the conserved mechanism of type VII ABC transporters and emphasizes the unique properties of LolCDE as a molecule extruder of triacylated lipoproteins.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Acilación , Adenosina Trifosfato/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Sitios de Unión , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Mutación , Periplasma/metabolismo , Conformación Proteica , Transporte de ProteínasRESUMEN
Cholesterol homeostasis and trafficking are critical to the maintenance of the asymmetric plasma membrane of eukaryotic cells. Disruption or dysfunction of cholesterol trafficking leads to numerous human diseases. ATP-binding cassette (ABC) transporters play several critical roles in this process, and mutations in these sterol transporters lead to disorders such as Tangier disease and sitosterolemia. Biochemical and structural information on ABC sterol transporters is beginning to emerge, with published structures of ABCA1 and ABCG5/G8; these two proteins function in the reverse cholesterol transport pathway and mediate the efflux of cholesterol and xenosterols to high-density lipoprotein and bile salt micelles, respectively. Although both of these transporters belong to the ABC family and mediate the efflux of a sterol substrate, they have many distinct differences. Here, we summarize the current understanding of sterol transport driven by ABC transporters, with an emphasis on these two extensively characterized transporters.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Transporte Biológico/fisiología , Esteroles/metabolismo , Animales , Colesterol/metabolismo , HumanosRESUMEN
Bedaquiline (BDQ, Sirturo) has been approved to treat multidrug resistant forms of Mycobacterium tuberculosis. Prior studies suggested that BDQ was a selective inhibitor of the ATP synthase from M. tuberculosis. However, Sirturo treatment leads to an increased risk of cardiac arrhythmias and death, raising the concern that this adverse effect results from inhibition at a secondary site. Here we show that BDQ is a potent inhibitor of the yeast and human mitochondrial ATP synthases. Single-particle cryo-EM reveals that the site of BDQ inhibition partially overlaps with that of the inhibitor oligomycin. Molecular dynamics simulations indicate that the binding mode of BDQ to this site is similar to that previously seen for a mycobacterial enzyme, explaining the observed lack of selectivity. We propose that derivatives of BDQ ought to be made to increase its specificity toward the mycobacterial enzyme and thereby reduce the side effects for patients that are treated with Sirturo.
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Diarilquinolinas/farmacología , Inhibidores Enzimáticos/farmacología , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Sitios de Unión , Microscopía por Crioelectrón , Diarilquinolinas/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Proteínas Fúngicas , Humanos , ATPasas de Translocación de Protón Mitocondriales/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Unión Proteica , Conformación Proteica , Reproducibilidad de los Resultados , Relación Estructura-ActividadRESUMEN
Mitochondria take up Ca2+ through the mitochondrial calcium uniporter complex to regulate energy production, cytosolic Ca2+ signalling and cell death1,2. In mammals, the uniporter complex (uniplex) contains four core components: the pore-forming MCU protein, the gatekeepers MICU1 and MICU2, and an auxiliary subunit, EMRE, essential for Ca2+ transport3-8. To prevent detrimental Ca2+ overload, the activity of MCU must be tightly regulated by MICUs, which sense changes in cytosolic Ca2+ concentrations to switch MCU on and off9,10. Here we report cryo-electron microscopic structures of the human mitochondrial calcium uniporter holocomplex in inhibited and Ca2+-activated states. These structures define the architecture of this multicomponent Ca2+-uptake machinery and reveal the gating mechanism by which MICUs control uniporter activity. Our work provides a framework for understanding regulated Ca2+ uptake in mitochondria, and could suggest ways of modulating uniporter activity to treat diseases related to mitochondrial Ca2+ overload.
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Canales de Calcio/química , Canales de Calcio/metabolismo , Microscopía por Crioelectrón , Sitios de Unión/efectos de los fármacos , Calcio/metabolismo , Calcio/farmacología , Canales de Calcio/ultraestructura , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructuraRESUMEN
Triacylglycerols store metabolic energy in organisms and have industrial uses as foods and fuels. Excessive accumulation of triacylglycerols in humans causes obesity and is associated with metabolic diseases1. Triacylglycerol synthesis is catalysed by acyl-CoA diacylglycerol acyltransferase (DGAT) enzymes2-4, the structures and catalytic mechanisms of which remain unknown. Here we determined the structure of dimeric human DGAT1, a member of the membrane-bound O-acyltransferase (MBOAT) family, by cryo-electron microscopy at approximately 3.0 Å resolution. DGAT1 forms a homodimer through N-terminal segments and a hydrophobic interface, with putative active sites within the membrane region. A structure obtained with oleoyl-CoA substrate resolved at approximately 3.2 Å shows that the CoA moiety binds DGAT1 on the cytosolic side and the acyl group lies deep within a hydrophobic channel, positioning the acyl-CoA thioester bond near an invariant catalytic histidine residue. The reaction centre is located inside a large cavity, which opens laterally to the membrane bilayer, providing lipid access to the active site. A lipid-like density-possibly representing an acyl-acceptor molecule-is located within the reaction centre, orthogonal to acyl-CoA. Insights provided by the DGAT1 structures, together with mutagenesis and functional studies, provide the basis for a model of the catalysis of triacylglycerol synthesis by DGAT.
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Biocatálisis , Microscopía por Crioelectrón , Diacilglicerol O-Acetiltransferasa/metabolismo , Diacilglicerol O-Acetiltransferasa/ultraestructura , Triglicéridos/biosíntesis , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Acilcoenzima A/ultraestructura , Aciltransferasas/química , Aciltransferasas/metabolismo , Dominio Catalítico , Membrana Celular/química , Membrana Celular/metabolismo , Diacilglicerol O-Acetiltransferasa/química , Histidina/química , Histidina/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Multimerización de Proteína , Especificidad por SustratoRESUMEN
ABCG2 is an ABC transporter that extrudes a variety of compounds from cells, and presents an obstacle in treating chemotherapy-resistant cancers. Despite recent structural insights, no anticancer drug bound to ABCG2 has been resolved, and the mechanisms of multidrug transport remain obscure. Such a gap of knowledge limits the development of novel compounds that block or evade this critical molecular pump. Here we present single-particle cryo-EM studies of ABCG2 in the apo state, and bound to the three structurally distinct chemotherapeutics. Without the binding of conformation-selective antibody fragments or inhibitors, the resting ABCG2 adopts a closed conformation. Our cryo-EM, biochemical, and functional analyses reveal the binding mode of three chemotherapeutic compounds, demonstrate how these molecules open the closed conformation of the transporter, and establish that imatinib is particularly effective in stabilizing the inward facing conformation of ABCG2. Together these studies reveal the previously unrecognized conformational cycle of ABCG2.
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Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Antineoplásicos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/ultraestructura , Antineoplásicos/química , Transporte Biológico , Disulfuros/metabolismo , Células HEK293 , Humanos , Mesilato de Imatinib/metabolismo , Ligandos , Mitoxantrona/química , Mitoxantrona/metabolismo , Modelos Biológicos , Estructura Secundaria de ProteínaRESUMEN
Gram-negative bacteria possess a dual-membrane envelope, which provides defense against environmental assault, as well as formidable resistance against antibiotics. Lipopolysaccharide (LPS) is the primary lipid component in the outermost membrane leaflet of most Gram-negative bacteria, and plays critical roles in cell envelope formation. Newly synthesized LPS at the cytoplasmic side of the inner membrane is flipped across the inner membrane and pushed across the periplasm by two ATP-binding cassette transporters, MsbA and LptB2FGC, respectively. Both transporters represent promising targets for developing new classes of antibiotics. In this review, we discuss recent advances in understanding the mechanism of LPS translocation driven by MsbA and LptB2FGC, with a particular focus on new findings from structural studies.