Sodium-dependent reorganization of the sugar-binding site of SGLT1

Bruce A. Hirayama, Donald D F Loo, Ana Díez-Sampedro, Daisy W. Leung, Anne Kristine Meinild, Mary Bing, Eric Turk, Ernest M. Wright

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)13391-13406
Number of pages16
JournalBiochemistry
Volume46
Issue number46
DOIs
StatePublished - Nov 20 2007

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Sugars
Deoxy Sugars
Sodium-Glucose Transport Proteins
Sodium
Binding Sites
Substrate Specificity
Hydroxyl Radical
Cysteine
Fluorescence
Ligands
Glucose
Conformations
Assays
Kinetics
Electric potential
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Hirayama, B. A., Loo, D. D. F., Díez-Sampedro, A., Leung, D. W., Meinild, A. K., Bing, M., ... Wright, E. M. (2007). Sodium-dependent reorganization of the sugar-binding site of SGLT1. Biochemistry, 46(46), 13391-13406. https://doi.org/10.1021/bi701562k

Sodium-dependent reorganization of the sugar-binding site of SGLT1. / Hirayama, Bruce A.; Loo, Donald D F; Díez-Sampedro, Ana; Leung, Daisy W.; Meinild, Anne Kristine; Bing, Mary; Turk, Eric; Wright, Ernest M.

In: Biochemistry, Vol. 46, No. 46, 20.11.2007, p. 13391-13406.

Research output: Contribution to journalArticle

Hirayama, BA, Loo, DDF, Díez-Sampedro, A, Leung, DW, Meinild, AK, Bing, M, Turk, E & Wright, EM 2007, 'Sodium-dependent reorganization of the sugar-binding site of SGLT1', Biochemistry, vol. 46, no. 46, pp. 13391-13406. https://doi.org/10.1021/bi701562k
Hirayama BA, Loo DDF, Díez-Sampedro A, Leung DW, Meinild AK, Bing M et al. Sodium-dependent reorganization of the sugar-binding site of SGLT1. Biochemistry. 2007 Nov 20;46(46):13391-13406. https://doi.org/10.1021/bi701562k
Hirayama, Bruce A. ; Loo, Donald D F ; Díez-Sampedro, Ana ; Leung, Daisy W. ; Meinild, Anne Kristine ; Bing, Mary ; Turk, Eric ; Wright, Ernest M. / Sodium-dependent reorganization of the sugar-binding site of SGLT1. In: Biochemistry. 2007 ; Vol. 46, No. 46. pp. 13391-13406.
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