19F NMR studies of the D-galactose chemosensory receptor. 2. Ca(II) binding yields a local structural change

The Escherichia coli D-galactose and D-glucose receptor possesses a Ca(II)-binding site closely related in structure and metal-binding characteristics to the eukaryotic EF-hand sites. Only the structure of the Ca(II)-occupied site is known. To investigate the structural change triggered by Ca(II) and Sr(II) binding, the authors have used 19F NMR to probe five 5-fluorotryptophan (5F-Trp) and seven 3-fluorophenylalanine (3F-Phe) positions in the structure. Of particular interest were two 5F-Trp residues near the N terminus of the Ca(II) site at positions 127 and 133 resonances, indicating a detectable structural change in the Ca(II) site. When the metal was removed from the Ca(II) site, the 5F-Trp127 and -133 frequencies shifted to a new value similar to that observed for free 5F-Trp in aqueous solvent, and this new frequency was a function of the H2O to D2O ratio, indicating that the residues had become solvent exposed. The allosteric coupling of the metal and sugar binding sites was observed to be slight. Together the results indicate that the structural changes in the Ca(II) site are primarily localized in the region of the site. Removal of the metal ion from the site exposes the nearby 5F-Trp127 and -133 residues to the solvent, suggesting that the empty site has a more open structure. Evidence for a similar opening of eukaryotic EF-hand sites to solvent upon removal of metal is discussed. Such a structural change could play an important role in facilitating substrate binding and dissociation