Structural Basis for the Mg2+-Dependent Modulation of Calmodulin Activity

Abstract

The physiological function of calmodulin (CaM) depends on its ability to respond specifically to micromolar Ca2+ signals in the presence of ∼1000-fold excess of the chemically similar Mg2+. The intracellular concentration of Mg2+ is tightly controlled in a narrow range of 0.5-1.0 mM, which is sufficient to fully or partially saturate the Ca2+-binding sites of many EF-hand proteins, including CaM, at the resting Ca2+ levels. To explain the mechanism of metal ion binding specificity we have compared the effects of Mg2+ (ionic radius r=0.72 A), Mn2+ (r=0.67 A) and Ca2+ (r=1.06 A) on the structure and stability of the N-terminal domain of calmodulin (N-CaM). The far UV-CD melting profiles show that all three metal ions stabilize the structure of N-CaM, as indicated by the concentration dependent increase in unfolding temperature from 52 °C (apo) to >100 °C, 95 °C and 75 °C in the presence of Ca2+, Mn2+ and Mg2+, respectively. We have determined the X-ray structures of N-CaM complexed with the three metal ions. Only the Ca2+-N-CaM complex features the open-domain active conformation, whereas Mg2+-N-CaM and Mn2+-N-CaM have a closed-domain, apo-like conformation. The relative positions of all bound metal ions with respect to the EF-hand-β-scaffold are similar, however the bidentate ligand, the Glu sidechain in the 12th position of the loop, interacts with Ca2+ only and does not bind directly to either Mn2+ or Mg2+. These results strongly support our proposal (Grabarek, BBA, 1813, 913, 2011) that the stereochemical constraints imposed by the two-EF-hand domain structure preclude Mg2+ from engaging the EF-hand ligands in the same way as Ca2+. Mg2+ stabilizes the apo-like conformation of an EF-hand, thus contributing to the switching off CaM activity at the resting Ca2+ concentrations.