Sodium-Dependent Reorganization of the Sugar-Binding Site of SGLT1
- 26 October 2007
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 46 (46), 13391-13406
- https://doi.org/10.1021/bi701562k
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.Keywords
This publication has 18 references indexed in Scilit:
- Molecular interactions between dipeptides, drugs and the human intestinal H+–oligopeptide cotransporter hPEPT1The Journal of Physiology, 2006
- Voltage Clamp Fluorometric Measurements on a Type II Na+-coupled Pi Cotransporter: Shedding Light on Substrate Binding OrderThe Journal of general physiology, 2006
- Residue 457 Controls Sugar Binding and Transport in the Na+/Glucose CotransporterPublished by Elsevier BV ,2001
- Purification and functional reconstitution of a truncated human Na+/glucose cotransporter (SGLT1) expressed inE. coliFEBS Letters, 1999
- State-dependent Accessibility and Electrostatic Potential in the Channel of the Acetylcholine ReceptorThe Journal of general physiology, 1998
- Membrane Topology Motifs in the SGLT Cotransporter FamilyThe Journal of Membrane Biology, 1997
- Five Transmembrane Helices Form the Sugar Pathway through the Na+/Glucose CotransporterPublished by Elsevier BV ,1997
- Compound missense mutations in the sodium/D-glucose cotransporter result in trafficking defectsGastroenterology, 1997
- Sugar Binding to Na+/Glucose Cotransporters Is Determined by the Carboxyl-terminal Half of the ProteinPublished by Elsevier BV ,1996
- Characterization of a Na+/glucose cotransporter cloned from rabbit small intestineThe Journal of Membrane Biology, 1989