Crystallographic characterization of a tri-Asp metal-binding site at the three-fold symmetry axis of LarE

Detailed crystallographic characterization of a tri-aspartate metal-binding site previously identified on the three-fold symmetry axis of a hexameric enzyme, LarE from Lactobacillus plantarum, was conducted. By screening an array of monovalent, divalent, and trivalent metal ions, we demonstrated that this metal binding site stoichiometrically binds Ca(2+), Mn(2+), Fe(2+)/Fe(3+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Cd(2+), but not monovalent metal ions, Cr(3+), Mg(2+), Y(3+), Sr(2+) or Ba(2+). Extensive database searches resulted in only 13 similar metal binding sites in other proteins, indicative of the rareness of tri-aspartate architectures, which allows for engineering such a selective multivalent metal ion binding site into target macromolecules for structural and biophysical characterization.