title: Sequences in the nonconsensus nucleotide-binding domain of ABCG5/ABCG8 required for sterol transport.
abstract: ATP-binding cassette transporters ABCG5 (G5) and ABCG8 (G8) form a heterodimer that transports cholesterol and plant sterols from hepatocytes into bile. Mutations that inactivate G5 or G8 cause hypercholesterolemia and premature atherosclerosis. We showed previously that the two nucleotide-binding domains (NBDs) in the heterodimer are not functionally equivalent; sterol transport is abolished by mutations in the consensus residues of NBD2 but not of NBD1. Here, we examined the structural requirements of NBD1 for sterol transport. Substitutions of the D-loop aspartate and Q-loop glutamine in either NBD did not impair sterol transport. The H-loop histidine of NBD2 (but not NBD1) was required for sterol transport. Exchange of the signature motifs between the NBDs did not interfere with sterol transport, whereas swapping the Walker A, Walker B, and signature motifs together resulted in failure to transport sterols. Selected substitutions within NBD1 altered substrate specificity: transport of plant sterols by the heterodimer was preserved, whereas transport of cholesterol was abolished. In summary, these data indicate that NBD1, although not required for ATP hydrolysis, is essential for normal function of G5G8 in sterol transport. Both the position and structural integrity of NBD2 are essential for sterol transport activity.
authors.affiliation: Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, Texas, 75390-8591, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.forename: Nick
authors.initials: N
authors.lastname: Grishin
authors.forename: Lisa
authors.initials: L
authors.lastname: Kinch
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Cohen
authors.forename: Helen H
authors.initials: HH
authors.lastname: Hobbs
authors.forename: Xiao-Song
authors.initials: XS
authors.lastname: Xie
mesh_terms: D000071018:ATP Binding Cassette Transporter, Subfamily G, Member 5; D000071019:ATP Binding Cassette Transporter, Subfamily G, Member 8; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D017124:Conserved Sequence; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008113:Liver Neoplasms; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D051381:Rats; D013261:Sterols / Q000378:metabolism*
chemical_list: C462600:ABCG5 protein, mouse; C462601:ABCG8 protein, mouse; D000071018:ATP Binding Cassette Transporter, Subfamily G, Member 5; D000071019:ATP Binding Cassette Transporter, Subfamily G, Member 8; D018528:ATP-Binding Cassette Transporters; D008074:Lipoproteins; D013261:Sterols; D002784:Cholesterol