Structural Analysis of Cholesterol Binding and Sterol Selectivity by ABCG5/G8.

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.

Structure of the Human Cholesterol Transporter ABCG1.

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.

Cryo-EM structure of ABCG5/G8 in complex with modulating antibodies.

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.

Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter.

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.

Picky ABCG5/G8 and promiscuous ABCG2 - a tale of fatty diets and drug toxicity.

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.

ABCG5/G8: a structural view to pathophysiology of the hepatobiliary cholesterol secretion.

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.

The E-helix is a central core in a conserved helical bundle involved in nucleotide binding and transmembrane domain intercalation in the ABC transporter superfamily.

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.

ABCG5 and ABCG8: more than a defense against xenosterols.

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.

Purification and biochemical characterization of NpABCG5/NpPDR5, a plant pleiotropic drug resistance transporter expressed in Nicotiana tabacum BY-2 suspension cells.

Pleiotropic drug resistance (PDR) transporters belong to the ABCG subfamily of ATP-binding cassette (ABC) transporters and are involved in the transport of various molecules across plasma membranes. During evolution, PDR genes appeared independently in fungi and in plants from a duplication of a half-size ABC gene. The enzymatic properties of purified PDR transporters from yeast have been characterized. This is not the case for any plant PDR transporter, or, incidentally, for any purified plant ABC transporter. Yet, plant PDR transporters play important roles in plant physiology such as hormone signaling or resistance to pathogens or herbivores. Here, we describe the expression, purification, enzymatic characterization and 2D analysis by electron microscopy of NpABCG5/NpPDR5 from Nicotiana plumbaginifolia, which has been shown to be involved in the plant defense against herbivores. We constitutively expressed NpABCG5/NpPDR5, provided with a His-tag in a homologous system: suspension cells from Nicotiana tabacum (Bright Yellow 2 line). NpABCG5/NpPDR5 was targeted to the plasma membrane and was solubilized by dodecyl maltoside and purified by Ni-affinity chromatography. The ATP-hydrolyzing specific activity (27 nmol min-1 mg-1) was stimulated seven-fold in the presence of 0.1% asolectin. Electron microscopy analysis indicated that NpABCG5/NpPDR5 is monomeric and with dimensions shorter than those of known ABC transporters. Enzymatic data (optimal pH and sensitivity to inhibitors) confirmed that plant and fungal PDR transporters have different properties. These data also show that N. tabacum suspension cells are a convenient host for the purification and biochemical characterization of ABC transporters.

Rice Stomatal Closure Requires Guard Cell Plasma Membrane ATP-Binding Cassette Transporter RCN1/OsABCG5.

Water stress is one of the major environmental stresses that affect agricultural production worldwide. Water loss from plants occurs primarily through stomatal pores. Here, we report that an Oryza sativa half-size ATP-binding cassette (ABC) subfamily G protein, RCN1/OsABCG5, is involved in stomatal closure mediated by phytohormone abscisic acid (ABA) accumulation in guard cells. We found that the GFP-RCN1/OsABCG5-fusion protein was localized at the plasma membrane in guard cells. The percentage of guard cell pairs containing both ABA and GFP-RCN1/OsABCG5 increased after exogenous ABA treatment, whereas they were co-localized in guard cell pairs regardless of whether exogenous ABA was applied. ABA application resulted in a smaller increase in the percentage of guard cell pairs containing ABA in rcn1 mutant (A684P) and RCN1-RNAi than in wild-type plants. Furthermore, polyethylene glycol (drought stress)-inducible ABA accumulation in guard cells did not occur in rcn1 mutants. Stomata closure mediated by exogenous ABA application was strongly reduced in rcn1 mutants. Finally, rcn1 mutant plants had more rapid water loss from detached leaves than the wild-type plants. These results indicate that in response to drought stress, RCN1/OsABCG5 is involved in accumulation of ABA in guard cells, which is indispensable for stomatal closure.