Phosphorylation at Ser26Ser^{26} in the ATP-Binding Site of Ca2+Ca^{2+}/Calmodulin-Dependent Kinase II as a Mechanism for Switching off the Kinase Activity

CaMKII $(Ca^{2+}$/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The $\gamma$ isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII $\gamma$ at $Ser^{26}$, a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at $Ser^{26}$, we generated a phosphospecific $Ser^{26}$ antibody and demonstrated an increase in $Ser^{26}$ phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of $Ser^{26}$ affects the kinase activity, we mutated $Ser^{26}$ to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in $Thr^{287}$ autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at $Ser^{26}$ of CaMKII $\gamma$ inhibits the catalytic activity of the enzyme towards its autophosphorylation site at $Thr^{287}$ most probably by blocking ATP binding. We propose that $Ser^{26}$ phosphorylation constitutes an important mechanism for switching off CaMKII activity

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 ∼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 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