Structural Consequences of Calmodulin EF Hand Mutations.

Calmodulin (CaM) is a cytosolic Ca ²⁺ -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 Ca ²⁺ , joined by a flexible central linker region. CaM is able to bind and activate its target proteins in the Ca ²⁺ -replete and Ca ²⁺ -deplete forms. To study the Ca ²⁺ -dependent/independent properties of binding and activation of target proteins by CaM, CaM constructs with Ca ²⁺ -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 Ca ²⁺ in either the N-lobe EF hands (CaM ₁₂ ), C-lobe EF hands (CaM ₃₄ ), or all four EF hands (CaM ₁₂₃₄ ). To investigate potential structural changes these mutations may cause, we performed detailed NMR studies of CaM ₁₂ , CaM ₃₄ , and CaM ₁₂₃₄ including determining the solution structure of CaM ₁₂₃₄ . 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 CaM ₃₄ bound to the iNOS CaM binding domain peptide. The structures provide direct structural evidence of changes that are present in these Ca ²⁺ -deficient CaM mutants and show these mutations increase the hydrophobic exposed surface and decrease the electronegative surface potential throughout each lobe of CaM. These Ca ²⁺ -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.