A direct test of the reductionist approach to structural studies of calmodulin activity: relevance of peptide models of target proteins

Ca(2+)-saturated calmodulin (CaM) directly associates with and activates CaM-dependent protein kinase I (CaMKI) through interactions with a short sequence in its regulatory domain. Using heteronuclear NMR (13)C-(15)N-(1)H correlation experiments, the backbone assignments were determined for CaM bound to a peptide (CaMKIp) corresponding to the CaM-binding sequence of CaMKI. A comparison of chemical shifts for free CaM with those of the CaM.CaMKIp complex indicate large differences throughout the CaM sequence. Using NMR techniques optimized for large proteins, backbone resonance assignments were also determined for CaM bound to the intact CaMKI enzyme. NMR spectra of CaM bound to either the CaMKI enzyme or peptide are virtually identical, indicating that calmodulin is structurally indistinguishable when complexed to the intact kinase or the peptide CaM-binding domain. Chemical shifts of CaM bound to a peptide (smMLCKp) corresponding to the calmodulin-binding domain of smooth muscle myosin light chain kinase are also compared with the CaM.CaMKI complexes. Chemical shifts can differentiate one complex from another, as well as bound versus free states of CaM. In this context, the observed similarity between CaM.CaMKI enzyme and peptide complexes is striking, indicating that the peptide is an excellent mimetic for interaction of calmodulin with the CaMKI enzyme.