Calcium(II) site specificity: effect of size and charge on metal ion binding to an EF-hand-like site.
The molecular mechanisms by which protein Ca(II) sites selectively bind Ca(II) even in the presence of high concentrations of other metals, particularly Na(I), K(I), and Mg(II), have not been fully described. The single Ca(II) site of the Escherichia coli receptor for D-galactose and D-glucose (GGR) is structurally related to the eukaryotic EF-hand Ca(II) sites and is ideally suited as a model for understanding the structural and electrostatic basis of Ca(II) specificity. Metal binding to the bacterial site was monitored by a Tb(III) phosphorescence assay: Ca(II) in the site was replaced with Tb(III), which was then selectively excited by energy transfer from protein tryptophans. Photons emitted from the bound Tb(III) enabled specific detection of this substrate; for other metals binding was detected by competitive displacement of Tb(III). Representative spherical metal ions from groups IA, IIA, and IIIA and the lanthanides were chosen to study the effects of metal ion size and charge on the affinity of metal binding. A dissociation constant was measured for each metal, yielding a range of KD's spanning over 6 orders of magnitude. Monovalent metal ions of group IA exhibited very low affinities. Divalent group IIA metal ions exhibited affinities related to their size, with optimal binding at an effective ionic radius between those of Mg(II) (0.81 A) and Ca(II) (1.06 A). Trivalent metal ions of group IIIA and the lanthanides also exhibited size-dependent affinities, with an optimal effective ionic radius between those of Sc(III) (0.81 A) and Yb(III) (0.925 A). The results indicate that the GGR site selects metal ions on the basis of both charge and size.(ABSTRACT TRUNCATED AT 250 WORDS)
Snyder
E E
EE
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Buoscio
B W
BW
Falke
J J
JJ
D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007763:Lac Operon; D008024:Ligands; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000235:genetics; D013816:Thermodynamics; D014867:Water / Q000378:metabolism
D002135:Calcium-Binding Proteins; D008024:Ligands; D011956:Receptors, Cell Surface; C039707:galactose receptor; C027786:glucose receptor; D014867:Water; D002118:Calcium