Cross-linking and fluorescence study of the COOH- and NH2-terminal domains of intact caldesmon bound to actin.

The NH2- and COOH-terminal domains of muscle caldesmon are separated by a long alpha-helical stretch. Cys-580, in the COOH-terminal domain, can be rapidly and efficiently disulfide-cross-linked to Cys-374 of actin by incubation with actin modified with 5,5'-dithiobis(2-nitrobenzoic acid) (Graceffa, P., and Jancso, A. (1991) J. Biol. Chem. 266, 20305-20310). Upon further incubation (+/- tropomyosin), a second cross-link was slowly formed between Cys-153 in the NH2-terminal domain and Cys-374 of another actin monomer. The yield of the second cross-link was relatively insensitive to increasing ionic strength, whereas the caldesmon-actin binding strength decreased considerably, suggesting that the NH2-terminal domain is largely dissociated from actin and becomes slowly cross-linked to it during collisions with the actin filament. In support of these conclusions, the yield of photocross-linking actin to caldesmon specifically labeled with benzophenonemaleimide at Cys-580 was high, but close to zero for caldesmon labeled at Cys-153, and the fluorescence intensity and polarization of tetramethylrhodamine iodoacetamide attached to Cys-580 showed large changes, while there were no changes for the probe at Cys-153 upon binding caldesmon to actin (+/- tropomyosin). These findings are consistent with the knowledge that COOH-terminal fragments of caldesmon bind to actin, whereas NH2-terminal fragments do not. Since the NH2-terminal domain of caldesmon binds to myosin, a dissociated NH2-terminal domain may account for caldesmon's ability to link myosin and actin filaments.