Structural Role of ABCG5/ABCG8 in Sterol Transport

Abstract

ATP binding cassette (ABC) transporters play critical roles in maintaining sterol homeostasis in eukaryotic organisms, including yeast, plants and mammals. In humans, the heterodimeric ABCG5/ABCG8 (G5G8) mediates the excretion of cholesterol and dietary plant sterols into bile and into the gut lumen. Mutations inactivating either ABCG5 or ABCG8 cause sitosterolemia, a rare autosomal recessive genetic disorder characterized by plant sterol accumulation, hypercholesterolemia, and premature coronary atherosclerosis. ABCG5 and ABCG8 are half ABC transporters;each subunit consists of an N-terminal nucleotide-binding domain (NBD) and a C-terminal transmembrane domain (TMD). The NBDs dimerize to form two catalytically asymmetric nucleotide-binding sites (NBS), one that is catalytically active (NBS2) and the other inactive (NBS1). To understand the structural basis for G5G8-mediated sterol transport we developed a large-scale purification of human G5G8 by exploiting Pichia patoris yeast. We crystallized the transporter in lipid bilayers, solved its structure in a nucleotide-free state at 3.9 Å resolution, and generated the first atomic model of an ABC sterol transporter. G5G8 presents a new structural configuration for the TMD of ABC transporters, which is present in a large and functionally diverse ABC2 superfamily. We discover that the TMD and the NBS are coupled through networks of interactions that differ between NBS1 and NBS2, reflecting the catalytic asymmetry of the transporter. A series of conserved polar residues in the TMD form polar networks that we proposed play a role in transmitting signals from the ATPase catalysis in the NBS to sterol transport on the TMD. Molecular dynamic simulation and long-range coevolution analysis revealed an inward-upward TMD movement that predicts a significant conformational change between the TMD subunits. Thus, the G5G8 structure provides a molecular framework that allows us to propose a mechanistic model for ABC transporter-mediated sterol transport and to analyze the disruptive effects of mutations causing sitosterolemia. The structure will serve as a structural template for homology modelling to a wide range of transport system that is regulated by ABCG transporters and by ABC2 superfamily.