X-ray Structures of Magnesium and Manganese Complexes with the N-Terminal Domain of Calmodulin: Insights into the Mechanism and Specificity of Metal Ion Binding to an EF-Hand

Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca2+ in the presence of an similar to 1000-fold higher concentration of cellular Mg2+. To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg2+, Mn2+, and Zn2+. In contrast to Ca2+, which induces domain opening in CaM, octahedrally coordinated Mg2+ and Mn2+ stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn2+ ions bind at the protein surface and do not compete with Ca2+. The relative positions of bound Mg2+ and Mn2+ within the EF-hand loops are similar to those of Ca2+; however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca2+ is critical for domain opening, does not bind directly to either Mn2+ or Mg2+, and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand beta-scaffold. The structures suggest that Mg2+ contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca2+. The Mg2+-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.