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

Abstract

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.

calcium-binding protein, calmodulin, conformation, evolution, yeast