THE CALMODULIN-BINDING DOMAIN OF CALDESMON BINDS TO CALMODULIN IN AN ALPHA-HELICAL CONFORMATION

The binding of calcium-calmodulin (CaM) to caldesmon (CaD) contributes to the regulation of smooth muscle contraction. It has been reported that a 17-residue synthetic peptide encompassing the residues Gly651-Ser667 of smooth muscle CaD constitutes its CaM-binding domain [Zhan, Q., Wong, S. S., and Wang, C. L. A. (1991) J. Biol. Chem. 266, 21810-21814]. This peptide does not share sequence homology with the CaM-binding domains of other proteins, and in addition, the binding of CaM to CaD is known to be relatively weak. Here we have investigated the properties of this atypical CaM-binding domain by NMR and circular dichroism (CD) spectroscopy. Two dimensional NMR studies performed in an aqueous TFE mixture (75%/25%) showed that the peptide has the capacity to adopt an amphiphilic alpha-helical conformation. TRNOESY experiments and CD spectroscopy were used to determine that the CaD peptide binds in an alpha-helical conformation to CaM. The addition of TFE or the binding of the CaD peptide to CaM induces an alpha-helical structure only for the central 10 amino acid residues of the peptide. Titrations of CaM with the CaD peptide were followed by proton NMR and show the formation of a 1:1 complex and that the binding is calcium-dependent. The chemical shifts of C-13-methyl groups of specifically labeled Met residues and of the N-15 backbone amide groups of CaM undergo changes upon addition of the CaD peptide; these data suggest that both domains and the central helix of CaM are involved in the binding of the peptide. A possible mode of binding of the CaD peptide to the methionine-rich regions of CaM, which is consistent with these data, is discussed.