Calmodulin binds to caldesmon in an antiparallel manner.

Abstract

Two of the five tryptophan residues (W659 and W692) in chicken gizzard smooth muscle caldesmon (CaD) are located within the calmodulin (CaM) binding sites in the C-terminal region of the molecule. When these Trp residues are replaced with Gly in either recombinant fragments or synthetic peptides of CaD, the affinity for CaM is decreased by at least 10-fold, suggesting that both of these residues are important for the interaction of CaD with CaM. To gain information about the topography of the CaM-CaD complex, we have carried out fluorescence titrations of CaM with Tb3+ as a substitute for Ca2+ in the presence of wild-type or mutated CaD variants. By exciting Trp residues of CaD fragments or peptides while monitoring the enhanced luminescence of CaM-bound Tb3+ ions via resonance energy transfer, we were able to estimate the relative proximity between the bound metal ions in the two domains of CaM and the Trp residues of CaD. Our results suggest that in the CaM-CaD complex the metal-binding sites III and IV in the C-terminal domain of CaM are very close to W659 of CaD; the N-terminal domain of CaM appears associated with the region of CaD in the vicinity of W692, although sites I and II are relatively far away from this Trp residue. These findings are consistent with a model in which CaM binds to CaD in an antiparallel manner. Such a binding mode, however, may be flexible enough to accommodate alternative spatial arrangements when the preferred binding sites are either altered or rendered unavailable.