title: Sodium-dependent reorganization of the sugar-binding site of SGLT1.
abstract: The sodium-dependent glucose cotransporter SGLT1 undergoes a series of voltage- and ligand-induced conformational changes that underlie the cotransport mechanism. In this study we describe how the binding of external Na changes the conformation of the sugar-binding domain, exposing residues that are involved in sugar recognition to the external environment. We constructed 15 individual Cys mutants in the four transmembrane helices (TMHs) that form the sugar binding and translocation domain. Each mutant was functionally characterized for transport kinetics and substrate specificity. Identification of interactions between mutated residues and hydroxyls on the pyranose ring was assessed by comparing the affinities of deoxy sugars to those of glucose. We determined conformation-dependent accessibility to the mutated residues by both a traditional substituted cysteine accessibility method (SCAM) and a new fluorescence binding assay. These data were integrated to orient the helices and construct a framework of residues that comprise the external sugar binding site. We present evidence that R499, Q457, and T460 play a direct role in sugar recognition and that five other residues are indirectly involved in transport. Arranging the four TMHs to account for Na-dependent accessibility and potential for sugar interaction allows us to propose a testable model for the SGLT1 sugar binding site.
authors.affiliation: Department of Physiology, The David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, Los Angeles, California 90095-1751, USA. bhirayama@mednet.ucla.edu
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Hirayama
authors.forename: Donald D F
authors.initials: DD
authors.lastname: Loo
authors.forename: Ana
authors.initials: A
authors.lastname: Díez-Sampedro
authors.forename: Daisy W
authors.initials: DW
authors.lastname: Leung
authors.forename: Anne-Kristine
authors.initials: AK
authors.lastname: Meinild
authors.forename: Mary
authors.initials: M
authors.lastname: Lai-Bing
authors.forename: Eric
authors.initials: E
authors.lastname: Turk
authors.forename: Ernest M
authors.initials: EM
authors.lastname: Wright
mesh_terms: D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012964:Sodium / Q000378:metabolism*; D051269:Sodium-Glucose Transporter 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014982:Xenopus laevis
chemical_list: C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; D012964:Sodium; D005947:Glucose; D003545:Cysteine