Structural Consequences of Calmodulin EF Hand Mutations

Calmodulin (CaM) is a cytosolic Ca²⁺-binding protein that serves as a control element for many enzymes. It consists of two globular domains, each containing two EF hand pairs capable of binding Ca²⁺, joined by a flexible central linker region. CaM is able to bind and activate its target proteins in the Ca²⁺-replete and Ca²⁺-deplete forms. To study the Ca²⁺-dependent/independent properties of binding and activation of target proteins by CaM, CaM constructs with Ca²⁺-binding disrupting mutations of Asp to Ala at position one of each EF hand have been used. These CaM mutant proteins are deficient in binding Ca²⁺ in either the N-lobe EF hands (CaM₁₂), C-lobe EF hands (CaM₃₄), or all four EF hands (CaM₁₂₃₄). To investigate potential structural changes these mutations may cause, we performed detailed NMR studies of CaM₁₂, CaM₃₄, and CaM₁₂₃₄ including determining the solution structure of CaM₁₂₃₄. We then investigated if these CaM mutants affected the interaction of CaM with a target protein known to interact with apoCaM by determining the solution structure of CaM₃₄ bound to the iNOS CaM binding domain peptide. The structures provide direct structural evidence of changes that are present in these Ca²⁺-deficient CaM mutants and show these mutations increase the hydrophobic exposed surface and decrease the electronegative surface potential throughout each lobe of CaM. These Ca²⁺-deficient CaM mutants may not be a true representation of apoCaM and may not allow for native-like interactions of apoCaM with its target proteins.