Intra- and Interdomain Effects Due to Mutation of Calcium-binding Sites in Calmodulin

The IQ-motif protein PEP-19, binds to the C-domain of calmodulin (CaM) with significantly different k_on and k_off rates in the presence and absence of Ca²⁺, which could play a role in defining the levels of free CaM during Ca²⁺ transients. The initial goal of the current study was to determine whether Ca²⁺ binding to sites III or IV in the C-domain of CaM was responsible for affecting the kinetics of binding PEP-19. EF-hand Ca²⁺-binding sites were selectively inactivated by the common strategy of changing Asp to Ala at the X-coordination position. Although Ca²⁺ binding to both sites III and IV appeared necessary for native-like interactions with PEP-19, the data also indicated that the mutations caused undesirable structural alterations as evidenced by significant changes in amide chemical shifts for apoCaM. Mutations in the C-domain also affected chemical shifts in the unmodified N-domain, and altered the Ca²⁺ binding properties of the N-domain. Conversion of Asp⁹³ to Ala caused the greatest structural perturbations, possibly due to the loss of stabilizing hydrogen bonds between the side chain of Asp⁹³ and backbone amides in apo loop III. Thus, although these mutations inhibit binding of Ca²⁺, the mutated CaM may not be able to support potentially important native-like activity of the apoprotein. This should be taken into account when designing CaM mutants for expression in cell culture.