Calmodulin mutant in central linker reduces the binding affinity with PreIQ and IQ while interacting with Ca1.2 channels.

Calmodulin (CaM) was reported to interact with PreIQ and IQ of Ca1.2 channels, but to date, no explicit binding sites of CaM were illustrated. Therefore, in the present study, we firstly used MOE (Molecular Operating Environment) for protein-protein docking and we found that the most likely residues of CaM that play an important role in the interface are concentrated in central linker region. Next we examined the binding properties of CaM and its mutants to PreIQ and IQ by GST pull-down assays. Here we confirmed that CaM binds to PreIQ and IQ in a concentration-dependent and [Ca]-dependent manner. However, silencing the effect of N-lobe and C-lobe by mutating two Ca binding sites of each lobe abolished [Ca]-dependence of CaM binding, but could not influence the combination. And the mutant in central linker reduced the binding of CaM/PreIQ and CaM/IQ especially at low [Ca]. We confirmed that N-lobe and C-lobe play vital role in sensing the change of Ca, and found that the central linker of CaM is involved in the binding of CaM to Ca1.2 channels in particular at low [Ca], not only participates in the combination with PreIQ, but also with IQ.