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
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Abstract
Calmodulin (CaM), a member of the EF-hand superfamily,
regulates
many aspects of cell function by responding specifically to micromolar
concentrations of Ca<sup>2+</sup> in the presence of an ∼1000-fold
higher concentration of cellular Mg<sup>2+</sup>. 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 Mg<sup>2+</sup>, Mn<sup>2+</sup>, and Zn<sup>2+</sup>. In contrast
to Ca<sup>2+</sup>, which induces domain opening in CaM, octahedrally
coordinated Mg<sup>2+</sup> and Mn<sup>2+</sup> stabilize the closed-domain,
apo-like conformation, while tetrahedrally coordinated Zn<sup>2+</sup> ions bind at the protein surface and do not compete with Ca<sup>2+</sup>. The relative positions of bound Mg<sup>2+</sup> and Mn<sup>2+</sup> within the EF-hand loops are similar to those of Ca<sup>2+</sup>; however, the Glu side chain at position 12 of the loop,
whose bidentate interaction with Ca<sup>2+</sup> is critical for domain
opening, does not bind directly to either Mn<sup>2+</sup> or Mg<sup>2+</sup>, 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 β-scaffold.
The structures suggest that Mg<sup>2+</sup> contributes to the switching
off of calmodulin activity and possibly other EF-hand proteins at
the resting levels of Ca<sup>2+</sup>. The Mg<sup>2+</sup>-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