title: The first nucleotide binding domain of the sulfonylurea receptor 2A contains regulatory elements and is folded and functions as an independent module.
abstract: The sulfonylurea receptor 2A (SUR2A) is an ATP-binding cassette (ABC) protein that forms the regulatory subunit of ATP-sensitive potassium (K(ATP)) channels in the heart. ATP binding and hydrolysis at the SUR2A nucleotide binding domains (NBDs) control gating of K(ATP) channels, and mutations in the NBDs that affect ATP hydrolysis and cellular trafficking cause cardiovascular disorders. To date, there is limited information on the SUR2A NBDs and the effects of disease-causing mutations on their structure and interactions. Structural and biophysical studies of NBDs, especially from eukaryotic ABC proteins like SUR2A, have been hindered by low solubility of the isolated domains. We hypothesized that the solubility of heterologously expressed SUR2A NBDs depends on the precise definition of the domain boundaries. Putative boundaries of SUR2A NBD1 were identified by structure-based sequence alignments and subsequently tested by exploring the solubility of SUR2A NBD1 constructs with different N and C termini. We have determined boundaries of SUR2A NBD1 that allow for soluble heterologous expression of the protein, producing a folded domain with ATP binding activity. Surprisingly, our alignment and screening data indicate that SUR2A NBD1 contains two putative, previously unidentified, regulatory elements: a large insert within the β-sheet subdomain and a C-terminal extension. Our approach, which combines the use of structure-based sequence alignments and predictions of disordered regions combined with biochemical and biophysical studies, may be applied as a general method for developing suitable constructs of other NBDs of ABC proteins.
authors.affiliation: Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada.
authors.forename: Elvin D
authors.initials: ED
authors.lastname: de Araujo
authors.forename: Lynn K
authors.initials: LK
authors.lastname: Ikeda
authors.forename: Svetlana
authors.initials: S
authors.lastname: Tzvetkova
authors.forename: Voula
authors.initials: V
authors.lastname: Kanelis
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054086:KATP Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D024661:Potassium Channels, Inwardly Rectifying / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D011955:Receptors, Drug / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D050436:Regulatory Elements, Transcriptional; D020218:Response Elements / Q000235:genetics; D012995:Solubility; D064233:Sulfonylurea Receptors
chemical_list: D018528:ATP-Binding Cassette Transporters; D004268:DNA-Binding Proteins; D054086:KATP Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; C092520:uK-ATP-1 potassium channel