Abstract
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.
Highlights
Smooth muscle contraction is primarily activated by myosin phosphorylation by a Ca2ϩ/calmodulin-dependent kinase and deactivated by dephosphorylation by a phosphatase [2]
Cross-linking and fluorescence experiments were performed at relatively high actin/caldesmon molar ratios (14 –20 for disulfide cross-linking and 20 for photocross-linking and fluorescence) in order to minimize any possible interference between caldesmon molecules on the actin filament, which might reduce the interaction between the caldesmon NH2-terminal domain and actin
Disulfide Cross-linking—Reaction between actin modified at Cys-374 with Nbs2 and gizzard caldesmon results in an almost complete disulfide cross-linking of caldesmon, via Cys-580 in the COOH-terminal domain, to actin Cys-374 in 2 min
Summary
Protein Preparations—Preparation of rabbit skeletal muscle actin and modification of actin at Cys-374 with 5,5Ј-dithiobis(2-nitrobenzoic acid) (Nbs2) were carried out as detailed earlier [29] except that the reaction was carried out at an Nbs2/actin molar ratio of 2.6 for 4 h at 0 °C. Caldesmon was extracted from the final pellet by resuspension in 4 volumes of extraction buffer (0.3 M NaCl, 50 mM Mops, pH 7.0, 1 mM EGTA, 0.5 mM MgCl2, 10 mM mercaptoethanol plus the above inhibitors), homogenized, and centrifuged as described above, and the supernatant was saved. The 30 – 50% saturated (NH4)2SO4 fraction of the supernatant was taken up in a small volume of 30 mM NaCl in DEAE buffer (0.2 mM EDTA, 10 mM Mops, pH 7.0, 0.1 mM DTT plus the above inhibitors) and dialyzed overnight against the same buffer. A high yield of the disulfide cross-link between Cys-580 of caldesmon and Cys-374 of actin was formed by incubating caldesmon with Nbs2-modified actin (Ϯtropomyosin) for 2 min at room temperature in buffer B (40 mM NaCl, 5 mM Mops, 2 mM MgCl2, pH 7.5, plus protease inhibitor mixture) (see Fig. 4 of Ref. 29 or Fig. 2A of this work). A cross-link between caldesmon Cys-153 and another actin Cys-374 formed slowly by further incubation for various times under these conditions or at other NaCl
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