Structural Consequences of Calmodulin EF Hand Mutations

Abstract

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.