Abstract
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.
Highlights
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
It is comprised of heterodimeric transmembrane domains (TMDs) and heterodimeric nucleotide-binding domains (NBD), which is responsible for ATP hydrolysis
For cryoelectron microscopic (cryo-EM) sample preparation, we purified the ternary complex of ABCG5/G8 with the Fab of both monoclonal antibodies (mAbs) 2E10 and 11F4 using size-exclusion chromatography and re-constituted the complex into saposin A-based nanodiscs before cryo-grid preparation and data collection (Fig. 1E and Supplementary Fig. 1)
Summary
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. Among the ABCG family members, ABCG5/G8 is essential for pumping cholesterol and phytosterols outward across apical membranes of enterocytes and hepatocytes[5] It serves as a gatekeeper of sterol transport and acts opposite to the Niemann–Pick C1-Like protein 1 (NPC1L1). With the availability of the crystal structure of ABCG5/G8, a comprehensive panel of human variants of each half transporter that alter transporter activity with different behaviors have been mapped to the structure[16] Key questions such as substrate specificity and ATP-driven cholesterol export remain unanswered. It remains unclear how the molecular mechanism operates whereby energy from ATP hydrolysis in the NBD is coupled to transport sterol substrates across cell membranes through the TMD.
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