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

Abstract

Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of the cell function by responding specifically to micromolar concentrations of Ca2+ in the presence of ~1000× higher concentration of cellular Mg2+. To explain the structural basis of metal ion binding specificity we have solved 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 sidechain in the 12th position 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-β-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.

Interaction with adenylate cyclase toxin from Bordetella pertussis affects the metal binding properties of calmodulin

“…The residues involved in Mg 2+ ligation and their corresponding chemical shift values were similar to those reported for free N‐terminal CaM 22. It has been shown that the smaller ionic radius of the Mg 2+ ion has different coordination geometry than the larger Ca 2+ , which limits the conformation plasticity in EF‐hand proteins 12, 22. Additionally, we found that residues mapping to sites I and II were perturbed in a way that is indicative of Mg 2+ ion coordination at each metal‐binding loop.…”

“…This is in direct contrast to TnC, which has monodentate ligation of the Mg 2+ ion mediated through Oε1 of Glu in the presence of target peptide 11, 30. TnC has been demonstrated to undergo a compaction in of the metal‐binding loops in the Mg 2+ ‐loaded state 11, whereas CaM does not coordinate Mg 2+ through the −Z ligand, so metal ligation is limited to the N‐terminal portion of the loop 12, 22. Metal binding specificity can be controlled by the amino acid side‐chain present at position 12 of the EF‐hand loop.…”

“…In Ca 2+ ‐loaded CaM, Oε1 and Oε2 of Glu residues coordinate the Ca 2+ ion in a bidentate fashion. However, in Mg 2+ ‐loaded CaM these side‐chain groups do not participate in Mg 2+ coordination 12, 22. This is in direct contrast to TnC, which has monodentate ligation of the Mg 2+ ion mediated through Oε1 of Glu in the presence of target peptide 11, 30.…”

“…Ca 2+ binding to sites I–IV promotes conformational rearrangements in CaM and subsequent exposure of hydrophobic surface areas in each domain, which is important in target peptide recognition 8. While Mg 2+ binding induces smaller, yet distinct structural transitions in CaM 9, 10, 11, 12, it is postulated to serve mainly as a suppressor of CaM‐dependent activation. In the presence of physiologically relevant Mg 2+ concentrations, it is reported that Ca 2+ affinity in CaM diminishes.…”

“…In the presence of physiologically relevant Mg 2+ concentrations, it is reported that Ca 2+ affinity in CaM diminishes. However, the Mg 2+ ‐binding constants in CaM are such that it is likely to be fully or partially Mg 2+ loaded at resting Ca 2+ concentrations 12. The probability of multiple intracellular populations consisting of Mg 2+ /Ca 2+ ‐loaded CaM conformers would suggest that a simple ‘on/off’ regulatory switch may not be adequate to control CaM‐dependent enzyme activation.…”