Quantitating and engineering the ion specificity of an EF-hand-like Ca2+ binding.
The Escherichia coli D-galactose and D-glucose receptor, an aqueous periplasmic receptor that triggers sugar sensing and transport, possesses a single Ca2+ binding site similar in structure and specificity to the EF-hand class of sites found in eukaryotic Ca2+ signaling proteins including calmodulin and its homologues. A universal feature of these sites is the use of a pentagonal bipyramidal array of seven oxygens to coordinate bound Ca2+. Here we investigate the mechanisms used by this coordinating array to control ion specificity. To vary the cavity size and charge of the array, we have replaced axial glutamine 142 in the prokaryotic site with asparagine, glutamate, and aspartate. The ion selectivities of the resulting engineered sites have been quantitated by measuring dissociation constants for a series of spherical metal ions, differing in increments of radius and charge, from groups Ia, IIa, and IIIa and the lanthanides. Dramatic specificity changes are observed: sites containing an engineered smaller side chain (Asn or Asp) bind the largest cations up to 50-fold more tightly than the native site; and sites containing an engineered negative side chain (Glu or Asp) exhibit preferences for trivalent over divalent cations up to 1900-fold higher than the native site. The results indicate that the cavity size and negative charge of the coordination array play key roles in selective Ca2+ binding and that the array can be engineered to preferentially bind other cations.
Falke
J J
JJ
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Snyder
E E
EE
Thatcher
K C
KC
Voertler
C S
CS
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D011487:Protein Conformation; D015202:Protein Engineering; D011956:Receptors, Cell Surface / Q000737:chemistry; D013816:Thermodynamics
D002135:Calcium-Binding Proteins; D011956:Receptors, Cell Surface; C039707:galactose receptor; C027786:glucose receptor; D002118:Calcium