A Sterol-Binding Cavity Underlies Sterol Recognition and Differential Activation of the ABCG5/G8

Sterol homeostasis depends on the coordinated regulation of endogenous cholesterol synthesis, dietary sterol absorption, and sterol excretion. The heterodimeric ATP-binding cassette sterol transporter ABCG5/G8 plays an important role in eliminating excess sterols by participating in reverse cholesterol transport and transintestinal cholesterol efflux. The molecular mechanism of sterol recognition and transport by ABCG5/G8 remains poorly understood. Here, we determined the cryo-electron microscopy (cryo-EM) structure of human ABCG5/G8 in complex with ergosterol. The structure revealed a sterol-binding site at the transmembrane domain (TMD) interface between the subunits ABCG5 and ABCG8, adjacent to the conserved aromatic clamp motif. Tyrosine 432 (Y432) on ABCG5, a key residue within the aromatic clamp, lies near the tetracyclic ring of ergosterol. Additionally, to assess the effect of different sterols on transporter activity, we performed molecular dynamic simulations and in vitro ATPase assays in the presence of cholesterol, cholesteryl hemisuccinate (CHS), and ergosterol. Ergosterol exhibited more favorable interactions with ABCG5/G8 and stimulated ATPase activity more effectively than either cholesterol or CHS, representing the first biochemical characterization of ABCG5/G8 activity in response to a non-cholesterol sterol. Furthermore, substitution of Y432 with the canonical phenylalanine in ABCG family abolished the differential ATPase response to ergosterol, with the mutant displaying similar activity levels in the presence of ergosterol and cholesterol. Together, our structural and biochemical findings reveal a conserved sterol-binding site within ABCG5/G8 and demonstrate direct evidence that distinct sterols differentially modulate ABCG sterol transporter activity and that the degenerative aromatic clamp motif in ABCG5 contributes to sterol-dependent functional selectivity.