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.