TRYPTOPHAN RESIDUES IN CALDESMON ARE MAJOR DETERMINANTS FOR CALMODULIN-BINDING

Calmodulin has been shown to interact with the COOH-terminal domain of gizzard h-caldesmon at three sites, A (residues 658-666), B (residues 687-695), and B' (residues 717-725), each of which contains a Trp res idue [Zhan et nl. (1991) J. Biol. Chem. 266, 21810-21814; Marston et a l. (1994) J. Biol. Chern. 269, 8134-8139: Mezgueldi er al. (1994) J. B iol. Chern. 269, 12824-12832]. To determine the contribution of each o f the three Trp residues in the calmodulin-caldesmon interaction, we h ave mutated the Trp residues to Ala in the COOH-terminal domain of fib roblast caldesmon (CaD39) and studied the effects on calmodulin bindin g by fluorescence measurements and using immobilized calmodulin. Wild- type CaD39 binds with a K-d of 0.13 x 10(-6) M and a stoichiometry of 1 mol of calmodulin per mol Replacing Trp 659 at site A or Trp 692 at site B to Ala reduces binding by 22- and 31 fold (K-d = 2.9 x 10(-6) a nd 4.0 x 10(-6) M), respectively, and destabilizes the CaD39-calmoduli n complex by 1.75 and 1.94 kcal mol(-1), respectively. Mutation of bot h Trp 659 and Trp 692 to Ala further reduces binding with a Kd of 6.1 x 10(-6) M and destabilizes the complex by 2.17 kcal mol(-1). On the o ther hand, mutation of Trp 722 at site B' to Ala causes a much smaller decrease in affinity (K-d = 0.6 x 10(6) M) and results in a destabili zation energy of 0.87 kcal mol(-1). To investigate the relative import ance of the amino acid residues near each Trp residue in the caldesmon -calmodulin interaction, deletion mutants were constructed lacking sit e A, site B, and site ASB. Although deletion of site A decreases bindi ng of CaD39 to calmodulin by 13-fold (K-d = 1.7 x 10(-6) M), it result s in tighter binding than mutation of Trp 659 to Ala at this site, sug gesting that the residues neighboring Trp 659 may contribute negativel y to the interaction. Deletion of site B causes a similar reduction in binding (K-d = 4.1 x 10(-6) M) as observed for replacing Trp 692 to A la at this site, indicating that Trp 692 is the major, if not the only , binding determinant at site B. Deletion of both site A and site B dr astically reduces binding by 62-fold. Taken together, these results su ggest that Trp 659 and Trp 692 are the major determinants in the calde smon-calmodulin interaction and that Trp 722 in site B' plays a minor role.