title: Functional hot spots in human ATP-binding cassette transporter nucleotide binding domains.
abstract: The human ATP-binding cassette (ABC) transporter superfamily consists of 48 integral membrane proteins that couple the action of ATP binding and hydrolysis to the transport of diverse substrates across cellular membranes. Defects in 18 transporters have been implicated in human disease. In hundreds of cases, disease phenotypes and defects in function can be traced to nonsynonymous single nucleotide polymorphisms (nsSNPs). The functional impact of the majority of ABC transporter nsSNPs has yet to be experimentally characterized. Here, we combine experimental mutational studies with sequence and structural analysis to describe the impact of nsSNPs in human ABC transporters. First, the disease associations of 39 nsSNPs in 10 transporters were rationalized by identifying two conserved loops and a small α-helical region that may be involved in interdomain communication necessary for transport of substrates. Second, an approach to discriminate between disease-associated and neutral nsSNPs was developed and tailored to this superfamily. Finally, the functional impact of 40 unannotated nsSNPs in seven ABC transporters identified in 247 ethnically diverse individuals studied by the Pharmacogenetics of Membrane Transporters consortium was predicted. Three predictions were experimentally tested using human embryonic kidney epithelial (HEK) 293 cells stably transfected with the reference multidrug resistance transporter 4 and its variants to examine functional differences in transport of the antiviral drug, tenofovir. The experimental results confirmed two predictions. Our analysis provides a structural and evolutionary framework for rationalizing and predicting the functional effects of nsSNPs in this clinically important membrane transporter superfamily.
authors.affiliation: Graduate Group in Bioinformatics, University of California at San Francisco, San Francisco, California, USA.
authors.forename: Libusha
authors.initials: L
authors.lastname: Kelly
authors.forename: Hisayo
authors.initials: H
authors.lastname: Fukushima
authors.forename: Rachel
authors.initials: R
authors.lastname: Karchin
authors.forename: Jason M
authors.initials: JM
authors.lastname: Gow
authors.forename: Leslie W
authors.initials: LW
authors.lastname: Chinn
authors.forename: Ursula
authors.initials: U
authors.lastname: Pieper
authors.forename: Mark R
authors.initials: MR
authors.lastname: Segal
authors.forename: Deanna L
authors.initials: DL
authors.lastname: Kroetz
authors.forename: Andrej
authors.initials: A
authors.lastname: Sali
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000225:Adenine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D063065:Organophosphonates / Q000737:chemistry / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D016415:Sequence Alignment; D000068698:Tenofovir
chemical_list: D018528:ATP-Binding Cassette Transporters; D000225:Adenine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D063065:Organophosphonates; D000068698:Tenofovir; C073492:ABCC4 protein, human
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