Specific conformation and Ca2+-binding mode of yeast calmodulin: insight into evolutionary development

The vertebrate calmodulin is configured with two structurally independent globular lobes in N- and C-terminus, and a flexible central linker. Distinctly, two lobes of calmodulin from Saccharomyces cerevisiae (yCaM) interact and influence the Ca2+-binding profile of each other. We explored this further using the mutant proteins with eliminated Ca2+-binding ability in one of the lobes and found that the Ca2+-bound N-lobe associates with the Ca2+-free C-lobe to gain the Ca2+ affinity of a wild-type level. Next, analysing series of C-terminal residue truncation mutant, we found that the truncation of C-terminal three residues induce the hyper Ca2+ affinity. These residues are also important for the general structural behaviour of calmodulin, such as Ca2+-induced slow mobility shift in polyacrylamide gel electrophoresis and for the ability to activate Cmk1p (yeast calmodulin kinase). These suggest: (i) when Ca2+ occupies only N-lobe, two lobes interact and form the stable intermediate leading to a proper level of Ca2+ affinity; (ii) the C-terminal three residues are required to prohibit abnormal stabilization of the intermediate promoting abnormally high Ca2+ affinity and for recognition of target enzymes. A model for Ca2+ and target bindings of yCaM is proposed. Evolutional aspect concerning the biological significance of this model was discussed.