Structural Studies of the Sterol Transporter ABCG5/G8: Nanodisc Reconstitution, Cryo-EM, and Nanobody Discovery

Abstract

ABCG5/G8 is a heterodimeric ATP binding cassette transporter that mediates cholesterol and dietary plant sterol efflux in the liver and intestine and plays an essential role in sterol homeostasis. Dysfunction of ABCG5/G8 is associated with sitosterolemia and cardiovascular disease risk. Despite progress in structural characterization, available structures have been determined in detergent micelles or restricted lipid environments which may not accurately reflect the native membrane context. In this thesis, ABCG5/G8 was expressed in Pichia pastoris, purified, and reconstituted into lipid nanodiscs by evaluating four membrane scaffold protein and lipid combinations. The optimal MSP1E3D1/POPC condition enabled single particle cryo-EM structure determination in a native-like lipid bilayer environment under apo, cholesterol-incubated, and cholesterol/AMP-PNP-incubated conditions, with the apo reconstruction reaching 3.15 Å resolution. In parallel, five unique ABCG5/G8 binding nanobodies were identified using yeast surface display. Together, this work establishes a foundation for structural studies of ABCG5/G8 in a native-like lipid bilayer environment and provides ABCG5/G8 specific nanobodies as new molecular tools for future structural and therapeutic applications.