Fluorine-19 NMR studies of the D-galactose chemosensory receptor. 1. Sugar binding yields a global structural change

Abstract

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.

Substrate‐induced conformational changes in Escherichia coli arginyl–tRNA synthetase observed by 19F NMR spectroscopy

“…2), so the reduction of the Trp172 sidechain may signi¢cantly a¡ect the inner structure of this domain, whereas the e¡ect on the local structure of Trp162 and Trp228 is over a longer distance. Similar perturbations of £uorine signals induced by Trp substitution have been previously observed in tissue factor [19], glucose and galactose binding protein [20], and leucine-speci¢c binding protein [21].…”

Dynamics of the S1S2 Glutamate Binding Domain of GluR2 Measured Using 19F NMR Spectroscopy

“…Full Agonists-19 F resonances are sensitive reporters of protein structure and dynamics (14). In particular, the 19 F chemical shift is very sensitive to changes in the electronic environment due to nearby hydrogen bonds, electrostatic fields, and van der Waals contacts (15), whereas the line-shape and relaxation parameters are good reporters of subtle changes in local dynamics.…”

THE TWO-COMPONENT SIGNALING PATHWAY OF BACTERIAL CHEMOTAXIS: A Molecular View of Signal Transduction by Receptors, Kinases, and Adaptation Enzymes

“…Cleft closure brings together the distinct docking patches on the two protein domains, thereby generating a contiguous docking surface. In addition, 19 F NMR studies of the galactose/glucose-binding protein reveal widespread conformational changes within both domains upon sugar binding to the inter-domain cleft, but not upon metal binding to a structural Ca 2+ -binding site (Luck & Falke 1991b,c, Danielson & Falke 1996. These findings raise the possibility that the global intradomain conformational rearrangments triggered specifically by the regulatory sugar ligand may contribute to the regulation of receptor docking surfaces, thereby augmenting the regulation provided by cleft closure.…”