Tryptophan Residues in Caldesmon Are Major Determinants for Calmodulin Binding

Abstract

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 residue [Zhan et al. (1991) J. Biol. Chem. 266, 21810-21814; Marston et al. (1994) J. Biol. Chem. 269, 8134-8139; Mezgueldi et al. (1994) J. Biol. Chem. 269, 12824-12832]. To determine the contribution of each of the three Trp residues in the calmodulin-caldesmon interaction, we have mutated the Trp residues to Ala in the COOH-terminal domain of fibroblast caldesmon (CaD39) and studied the effects on calmodulin binding by fluorescence measurements and using immobilized calmodulin. Wild-type CaD39 binds with a K d of 0.13 × 10 -6 M and a stoichiometry of 1 mol of calmodulin per mol of caldesmon. 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 × 10 -6 and 4.0 × 10 -6 M), respectively, and destabilizes the CaD39-calmodulin complex by 1.75 and 1.94 kcal mol -1 , respectively. Mutation of both Trp 659 and Trp 692 to Ala further reduces binding with a K d of 6.1 × 10 -6 M and destabilizes the complex by 2.17 kcal mol -1 . On the other hand, mutation of Trp 722 at site B′ to Ala causes a much smaller decrease in affinity (K d ) 0.6 × 10 -6 M) and results in a destabilization energy of 0.87 kcal mol -1 . To investigate the relative importance of the amino acid residues near each Trp residue in the caldesmon-calmodulin interaction, deletion mutants were constructed lacking site A, site B, and site A+B. Although deletion of site A decreases binding of CaD39 to calmodulin by 13-fold (K d ) 1.7 × 10 -6 M), it results in tighter binding than mutation of Trp 659 to Ala at this site, suggesting that the residues neighboring Trp 659 may contribute negatively to the interaction. Deletion of site B causes a similar reduction in binding (K d ) 4.1 × 10 -6 M) as observed for replacing Trp 692 to Ala 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 drastically reduces binding by 62-fold. Taken together, these results suggest that Trp 659 and Trp 692 are the major determinants in the caldesmoncalmodulin interaction and that Trp 722 in site B′ plays a minor role.

Caldesmon mutant defective in Ca2+-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility

“…Binding ability of CaD39-AB to Ca 2+ -CaM is greatly reduced by the mutation It was shown previously that two Trp residues at the C-terminal fragment of human fibroblast CaD (CaD39), W460 and W494, were crucial for Ca 2+ -CaM binding (Graether et al, 1997). Using site-directed mutagenesis, a mutant of the CaD39 fragment was generated in which both W460 and W494 were substituted with alanine, and named CaD39-AB (Fig.…”

“…In human smooth muscle CaD, site A and site B localized between amino acids 715-722 and 744-752, respectively (Hayashi et al, 1992;Humphrey et al, 1992). It has been shown that the tryptophan residues within the two domains are crucial for Ca 2+ -CaM binding (Graether et al, 1997). Despite a wealth of in vitro evidence, there is very little evidence suggesting that CaD activity is indeed regulated by Ca 2+ -CaM in living cells or that it plays a role in controlling actin filament dynamics.…”

Caldesmon is an integral component of podosomes in smooth muscle cells

“…We have shown before that Trp454 and Trp487, at calmodulin-binding sites A and B, respectively, are essential for caldesmon to interact with calmodulin; mutation of these Trp residues to Ala abolished the caldesmon-calmodulin Journal of Cell Science 119 (9) interaction (Graether et al, 1997). Overexpression of the fulllength l-caldesmon containing the W454A and W487A mutations (CadCamAB) disrupted the normally straight and parallel array of stress fibres as well as focal adhesions.…”

“…The C-terminal fragment of lcaldesmon (Cad39; amino acids 236-532) contains two tropomyosin-binding sites, two actin-interacting regions (diamonds) (Wang et al, 1997b;Marston et al, 1998), and the two Ca 2+ /calmodulin binding sites A and B, as indicated (dark-grey boxes) Wang et al, 1996). Both Ca 2+ /calmodulin-binding sites contain a key tryptophan residue (W454 and W487) that is essential for this interaction (Graether et al, 1997); replacement of these tryptophan residues with alanine (W454A and W487A) generates an l-caldesmon mutant that is unable to interact with Ca 2+ /calmodulin (CadCamAB).…”

“…We generated an EGFP-caldesmon construct (CadCamAB), in which the two tryptophan residues, W454 and W487, known to be essential for calmodulin-binding were mutated to Ala. The W454A and W487A mutations have been shown to abolish interaction between caldesmon and calmodulin, but have little effect on binding of caldesmon to actin (Graether et al, 1997;Wang et al, 1997b;Li et al, 2004). Depending on the expression level, CadCamAB caused various degrees of stress fibre disassembly.…”