RESUMEN
The ATP-binding cassette (ABC) sterol transporters are responsible for maintaining cholesterol homeostasis in mammals by participating in reverse cholesterol transport (RCT) or transintestinal cholesterol efflux (TICE). The heterodimeric ABCG5/G8 carries out selective sterol excretion, preventing the abnormal accumulation of plant sterols in human bodies, while homodimeric ABCG1 contributes to the biogenesis and metabolism of high-density lipoproteins. A sterol-binding site on ABCG5/G8 was proposed at the interface of the transmembrane domain and the core of lipid bilayers. In this study, we have determined the crystal structure of ABCG5/G8 in a cholesterol-bound state. The structure combined with amino acid sequence analysis shows that in the proximity of the sterol-binding site, a highly conserved phenylalanine array supports functional implications for ABCG cholesterol/sterol transporters. Lastly, in silico docking analysis of cholesterol and stigmasterol (a plant sterol) suggests sterol-binding selectivity on ABCG5/G8, but not ABCG1. Together, our results provide a structural basis for cholesterol binding on ABCG5/G8 and the sterol selectivity by ABCG transporters.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5 , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8 , Colesterol , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/genética , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/química , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/genética , Colesterol/química , Colesterol/metabolismo , Microscopía por Crioelectrón , Humanos , Membrana Dobles de Lípidos/química , Lipoproteínas HDL/metabolismo , Fenilalanina/metabolismo , Fitosteroles/metabolismo , Unión Proteica , Conformación Proteica , Estigmasterol/metabolismoRESUMEN
The ABCG1 homodimer (G1) and ABCG5-ABCG8 heterodimer (G5G8), two members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter G family, are required for maintenance of cellular cholesterol levels. G5G8 mediates secretion of neutral sterols into bile and the gut lumen, whereas G1 transports cholesterol from macrophages to high-density lipoproteins (HDLs). The mechanisms used by G5G8 and G1 to recognize and export sterols remain unclear. Here, we report cryoelectron microscopy (cryo-EM) structures of human G5G8 in sterol-bound and human G1 in cholesterol- and ATP-bound states. Both transporters have a sterol-binding site that is accessible from the cytosolic leaflet. A second site is present midway through the transmembrane domains of G5G8. The Walker A motif of G8 adopts a unique conformation that accounts for the marked asymmetry in ATPase activities between the two nucleotide-binding sites of G5G8. These structures, along with functional validation studies, provide a mechanistic framework for understanding cholesterol efflux via ABC transporters.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/metabolismo , Adenosina Trifosfato/metabolismo , Colesterol/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/genética , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/química , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/genética , Sitios de Unión , Transporte Biológico , Microscopía por Crioelectrón , Humanos , Conformación ProteicaRESUMEN
ABCG1 is an ATP binding cassette (ABC) transporter that removes excess cholesterol from peripheral tissues. Despite its role in preventing lipid accumulation and the development of cardiovascular and metabolic disease, the mechanism underpinning ABCG1-mediated cholesterol transport is unknown. Here we report a cryo-EM structure of human ABCG1 at 4 Å resolution in an inward-open state, featuring sterol-like density in the binding cavity. Structural comparison with the multidrug transporter ABCG2 and the sterol transporter ABCG5/G8 reveals the basis of mechanistic differences and distinct substrate specificity. Benzamil and taurocholate inhibited the ATPase activity of liposome-reconstituted ABCG1, whereas the ABCG2 inhibitor Ko143 did not. Based on the structural insights into ABCG1, we propose a mechanism for ABCG1-mediated cholesterol transport.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/química , Adenosina Trifosfato/química , Amilorida/análogos & derivados , Colesterol/química , Proteínas de Neoplasias/química , Ácido Taurocólico/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/metabolismo , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/antagonistas & inhibidores , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/química , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/genética , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/metabolismo , Adenosina Trifosfato/metabolismo , Amilorida/química , Amilorida/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Colesterol/metabolismo , Microscopía por Crioelectrón , Dicetopiperazinas/química , Dicetopiperazinas/farmacología , Expresión Génica , Células HEK293 , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Cinética , Lipoproteínas/antagonistas & inhibidores , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Ácido Taurocólico/químicaRESUMEN
The heterodimer of ATP-binding cassette transporter ABCG5 and ABCG8 mediates the excretion of sterols from liver and intestine, playing a critical role in cholesterol homeostasis. Here, we present the cryo-EM structure of ABCG5/G8 in complex with the Fab fragments from two monoclonal antibodies at 3.3Å resolution. The high-resolution structure reveals a unique dimer interface between the nucleotide-binding domains (NBD) of opposing transporters, consisting of an ordered network of salt bridges between the conserved NPXDFXXD motif and serving as a pivot point that may be important for the transport cycle. While mAb 11F4 increases the ATPase activity potentially by stabilization of the NBD dimer formation, mAb 2E10 inhibits ATP hydrolysis, likely by restricting the relative movement between the RecA and helical domain of ABCG8 NBD. Our study not only provides insights into the structural elements important for the transport cycle but also reveals novel epitopes for potential therapeutic interventions.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/metabolismo , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/química , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Microscopía por Crioelectrón/métodos , Lipoproteínas/química , Lipoproteínas/metabolismo , Adenosina Trifosfato/metabolismo , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/metabolismo , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/metabolismo , Adenosina Trifosfatasas/metabolismo , Ésteres del Colesterol/metabolismo , Colesterol/metabolismo , Ácido Cólico/metabolismo , Lipoproteínas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/genética , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/química , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/genética , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Sitios de Unión , Transporte Biológico , Colesterol/química , Ésteres del Colesterol/química , Ácido Cólico/química , Expresión Génica , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patología , Enfermedades Intestinales/genética , Enfermedades Intestinales/metabolismo , Enfermedades Intestinales/patología , Cinética , Errores Innatos del Metabolismo Lipídico/genética , Errores Innatos del Metabolismo Lipídico/metabolismo , Errores Innatos del Metabolismo Lipídico/patología , Lipoproteínas/química , Lipoproteínas/genética , Modelos Moleculares , Mutación , Fitosteroles/efectos adversos , Fitosteroles/genética , Fitosteroles/metabolismo , Pichia/química , Pichia/genética , Pichia/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinámicaRESUMEN
Structural data on ABCG5/G8 and ABCG2 reveal a unique molecular architecture for subfamily G ATP-binding cassette (ABCG) transporters and disclose putative substrate-binding sites. ABCG5/G8 and ABCG2 appear to use several unique structural motifs to execute transport, including the triple helical bundles, the membrane-embedded polar relay, the re-entry helices, and a hydrophobic valve. Interestingly, ABCG2 shows extreme substrate promiscuity, whereas ABCG5/G8 transports only sterol molecules. ABCG2 structures suggest a large internal cavity, serving as a binding region for substrates and inhibitors, while mutational and pharmacological analyses support the notion of multiple binding sites. By contrast, ABCG5/G8 shows a collapsed cavity of insufficient size to hold substrates. Indeed, mutational analyses indicate a sterol-binding site at the hydrophobic interface between the transporter and the lipid bilayer. In this review, we highlight key differences and similarities between ABCG2 and ABCG5/G8 structures. We further discuss the relevance of distinct and shared structural features in the context of their physiological functions. Finally, we elaborate on how ABCG2 and ABCG5/G8 could pave the way for studies on other ABCG transporters.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/metabolismo , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/metabolismo , Dieta , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Preparaciones Farmacéuticas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/genética , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/química , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/genética , Animales , Evolución Molecular , Humanos , Modelos Moleculares , Especificidad por SustratoRESUMEN
The ABCG5/G8 heterodimer is the primary neutral sterol transporter in hepatobiliary and transintestinal cholesterol excretion. Inactivating mutations on either the ABCG5 or ABCG8 subunit cause Sitosterolemia, a rare genetic disorder. In 2016, a crystal structure of human ABCG5/G8 in an apo state showed the first structural information on ATP-binding cassette (ABC) sterol transporters and revealed several structural features that were observed for the first time. Over the past decade, several missense variants of ABCG5/G8 have been associated with non-Sitosterolemia lipid phenotypes. In this review, we summarize recent pathophysiological and structural findings of ABCG5/G8, interpret the structure-function relationship in disease-causing variants and describe the available evidence that allows us to build a mechanistic view of ABCG5/G8-mediated sterol transport.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/química , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/química , Sistema Biliar/metabolismo , Colesterol/metabolismo , Lipoproteínas/química , Hígado/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/biosíntesis , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/metabolismo , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/biosíntesis , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/metabolismo , Animales , Catálisis , Homeostasis , Humanos , Hipercolesterolemia/metabolismo , Enfermedades Intestinales/metabolismo , Errores Innatos del Metabolismo Lipídico/metabolismo , Lipoproteínas/biosíntesis , Lipoproteínas/metabolismo , Fitosteroles/efectos adversos , Fitosteroles/metabolismoRESUMEN
ABC transporter proteins are involved in active transport, both in prokaryotes and eukaryotes. Sequence analysis of nucleotide binding domains (NBDs) of ABC proteins from all taxa revealed a well-conserved new motif having the signature: xT/ShxE/DNhxF, located between Q-loop and ABC signature sequence. A recent structure of an ABC transporter, ABCG5/G8 highlighted the motif as an essential structural determinant of inter-domain crosstalk and termed it as E-helix. We carried out an extensive computational analysis to unravel important structural entities alongside E-helix which plausibly play role in the interlocking mechanism of NBD with TMD. We identified E-helix to be a central structural moiety which interacts with three helices and an intracellular loop that leads to the transmembrane domain. Considering its wide occurrence, we examined the importance of this motif in one representative multidrug ABC transporter of Candida albicans, Cdr1p. The motif residues were replaced by alanines both individually as well as in combinations. The GFP-tagged versions of mutant proteins were overexpressed in Saccharomyces cerevisiae. Overall, our mutational data suggested that this motif plays a role in the maintenance of proper structural fold and/or inter-domain contacts in Cdr1p. We, thus, unveil an essential structural motif in ABC superfamily transporters.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/química , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/química , Transportadoras de Casetes de Unión a ATP/química , Proteínas Fúngicas/química , Lipoproteínas/química , Proteínas de Transporte de Membrana/química , Proteínas de Saccharomyces cerevisiae/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/genética , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/genética , Transportadoras de Casetes de Unión a ATP/genética , Secuencias de Aminoácidos , Animales , Sitios de Unión , Proteínas Fúngicas/genética , Humanos , Lipoproteínas/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Relación Estructura-ActividadRESUMEN
The elucidation of the molecular basis of the rare disease, sitosterolemia, has revolutionized our mechanistic understanding of how dietary sterols are excreted and how cholesterol is eliminated from the body. Two proteins, ABCG5 and ABCG8, encoded by the sitosterolemia locus, work as obligate dimers to pump sterols out of hepatocytes and enterocytes. ABCG5/ABCG8 are key in regulating whole-body sterol trafficking, by eliminating sterols via the biliary tree as well as the intestinal tract. Importantly, these transporters keep xenosterols from accumulating in the body. The sitosterolemia locus has been genetically associated with lipid levels and downstream atherosclerotic disease, as well as formation of gallstones and the risk of gallbladder cancer. While polymorphic variants raise or lower the risks of these phenotypes, loss of function of this locus leads to more dramatic phenotypes, such as premature atherosclerosis, platelet dysfunction, and thrombocytopenia, and, perhaps, increased endocrine disruption and liver dysfunction. Whether small amounts of xenosterol exposure over a lifetime cause pathology in normal humans with polymorphic variants at the sitosterolemia locus remains largely unexplored. The purpose of this review will be to summarize the current state of knowledge, but also highlight key conceptual and mechanistic issues that remain to be explored.