Transmembrane polar relay drives the allosteric regulation for ABCG5/G8 sterol transporter

Abstract

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 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 ATP catalysis by using three loss-of-function missense variants, two sitosterolemia mutations (E146Q and R543S; within polar relay) and one sterol-binding mutation (A540F; 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 polar relay network by all three mutants. Our data herein provide a biochemical evidence underlying the importance of the polar relay in regulating the catalytic activity of ABCG5/G8 sterol transporter.

Structural analysis of cholesterol binding and sterol selectivity by ABCG5/G8

“…By exploiting the LOF mutant A540F, in vivo and in vitro functional studies support the notion that it is exposed to the orthogonal cholesterol-binding site [25, 29] ( Fig. 2 ).…”

“…2 ). A mutant ABCG5 A540F was previously demonstrated to downregulate biliary cholesterol efflux in vivo [25] and to inhibit sterol-coupled ATPase activity in vitro [29]. The crystal structure herein provides a structural evidence of this peripheral sterol binding site where a sterol molecule makes direct contact with the conserved ABCG5 A540 residue ( Fig.…”

“…With an in vivo analysis of biliary cholesterol secretion and an in vitro ATPase assay, we have previously characterized a loss-of-function (LOF) mutant ABCG5 A540F /G8 (A540F), predicting a putative sterol-binding at this transporter-membrane interface [25, 29]. In this study, we developed an in vitro cholesterol-binding assay using tritiated cholesterol (see Materials and Methods) and characterized cholesterol binding on purified ABCG5/G8 WT and A540F mutant proteins ( Fig.…”

“…The binding constant K D and Hill coefficient h were estimated as 1.46 ± 0.28 μM (h = 2.91) and 1.55 ± 0.01 μM (h = 2.98) for the WT and mutant proteins, respectively. Given the ATPase activity of A540F mutation was suppressed by ∼90% with similar K M for sterols [29], both assays suggest a functionally defective conformation by this LOF mutant. Together with the new structural data, our analysis supports the role of the orthogonal cholesterol-binding site in proximity to ABCG5 A540 ( Fig.…”

“…Such revisions may come from the subduing of lipid rafts by redistributing the high cholesterol content towards the surrounding membrane regions [16, 28]. Using in vivo and in vitro functional assays, we have previously characterized a loss-of-function (LOF) mutation ABCG5 A540F , a conserved residue near a putative sterol-binding site in ABCG5/G8 [25, 29]. These findings suggested the likelihood of an access site that allows the binding of cholesterol binding prior to its re-localization within the membrane-embedded portion of the sterol transporters.…”