Calcium-dependent and -independent interactions of the calmodulin-binding domain of cyclic nucleotide phosphodiesterase with calmodulin

The ubiguitous Ca2+-binding regulatory protein calmodulin (CaM) binds and a ctivates a wide range of regulatory enzymes. The: binding is usually depend ent on the binding of Ca2+ to CaM; however, some target proteins interact w ith CaM in a calcium-independent manner. In this work, we have studied the interactions between CaM and a 20-residue synthetic peptide encompassing th e major calmodulin-binding domain of cyclic nucleotide phosphodiesterase (P DE1A2). The binding was studied in the absence and presence of Ca2+. by far -UV and near-UV circular dichroism, fluorescence, and infrared spectroscopy . In addition, two-dimensional heteronuclear NMR studies with C-13-methyl-M et-CaM and uniformly N-15- labeled CaM were performed. Competition assays w ith smooth muscle myosin light chain kinase revealed a K-d of 224 nM for pe ptide binding to Ca2+-CaM, while binding of the peptide to apo-CaM is weake r. The peptide binds with an alpha-helical structure to both lobes of Ca2+- saturated CaM, and the single Trp residue is firmly anchored into the C-ter minal lobe of CaM. In contrast, the Trp residue plays a minor role in the b inding to the ape-protein. Moreover, when bound to apo-CaM, the PDE peptide is only partially helical, and it interacts solely with the C-terminal lob e of CaM. These results show that the Ca2+-induced activation of PDE involv es a significant change in the structure and positioning of the CaM-bound P DE peptide domain.