19F NMR studies of the D-galactose chemosensory receptor. 1. Sugar binding yields a global structural change.
The Escherichia coli D-galactose and D-glucose receptor is an aqueous sugar-binding protein and the first component in the distinct chemosensory and transport pathways for these sugars. Activation of the receptor occurs when the sugar binds and induces a conformational change, which in turn enables docking to specific membrane proteins. Only the structure of the activated receptor containing bound D-glucose is known. To investigate the sugar-induced structural change, we have used 19F NMR to probe 12 sites widely distributed in the receptor molecule. Five sites are tryptophan positions probed by incorporation of 5-fluorotryptophan; the resulting 19F NMR resonances were assigned by site-directed mutagenesis. The other seven sites are phenylalanine positions probed by incorporation of 3-fluorophenylalanine. Sugar binding to the substrate binding cleft was observed to trigger a global structural change detected via 19F NMR frequency shifts at 10 of the 12 labeled sites. Two of the altered sites lie in the substrate binding cleft in van der Waals contact with the bound sugar molecule. The other eight altered sites, specifically two tryptophans and six phenylalanines distributed equally between the two receptor domains, are distant from the cleft and therefore experience allosteric structural changes upon sugar binding. The results are consistent with a model in which multiple secondary structural elements, known to extend between the substrate cleft and the protein surface, undergo shifts in their average positions upon sugar binding to the cleft. Such structural coupling provides a mechanism by which sugar binding to the substrate cleft can cause structural changes at one or more docking sites on the receptor surface.
Luck
L A
LA
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Falke
J J
JJ
D050260:Carbohydrate Metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005461:Fluorine; D015964:Gene Expression Regulation, Bacterial; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D010957:Plasmids; D011956:Receptors, Cell Surface / Q000737:chemistry*
D011956:Receptors, Cell Surface; C039707:galactose receptor; C027786:glucose receptor; D005461:Fluorine