Closer proximity between opposing domains of vertebrate calmodulin following deletion of Met(145)-Lys(148).

Abstract

To investigate the structural linkage between the opposing globular domains in vertebrate calmodulin (CaM), we have constructed a CaM mutant (CaMX(145)) deficient in the last four amino acids between Met(145) and Lys(148) at the carboxyl terminal. Circular dichroism and fluorescence spectroscopic measurements were used to detect changes in the average secondary and tertiary structure of CaMX(145) in comparison to full-length CaM. Complementary measurements of the maximal calcium-binding stoichiometry and ability to activate the plasma membrane (PM) Ca-ATPase permit an assessment of the functional significance of observed structural changes. In comparison with native CaM, we find that CaMX(145) exhibits (i) a large reduction in alpha-helical content, (ii) a dramatic decrease in the average spatial separation between the opposing globular domains, (iii) the loss of one high-affinity calcium-binding site, and (iv) a diminished binding affinity for the PM-Ca-ATPase. Thus, the sequence near the carboxyl terminus functions to stabilize high-affinity calcium binding at one site and facilitates important intramolecular interactions that maintain CaM in an extended conformation. However, despite the large conformational changes resulting from deletion of the last four amino acids at the carboxyl terminal, CaMX(145) can fully activate the PM-Ca-ATPase. These results indicate that target protein binding can restore the nativelike structure critical to function, emphasizing that the structure of the central helix is not critical to CaM function under equilibrium conditions. Rather, the central helix functions to maintain the spatial separation between the opposing domains in CaM that may be critical to high-affinity binding and the rapid activation of the PM-Ca-ATPase, which are necessary for optimal calcium signaling. Thus, following initial association between CaM and target proteins, structural changes involving the carboxyl-terminal sequence have the potential to play an important role in triggering the structural collapse of CaM that facilitates the rapid and cooperative binding of the opposing globular domains with target proteins, which is important to high-affinity binding and rapid enzyme activation.