Location and functional characterization of myosin contact sites in smooth- muscle caldesmon
| Autor: | B. Steven Marston, A. J. Pia Huber, V. Alexander Vorotnikov |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
animal structures
Myosin light-chain kinase Macromolecular Substances macromolecular substances Myosins Binding Competitive Biochemistry Divalent Mole Myosin Animals Binding site Molecular Biology Actin chemistry.chemical_classification biology Myosin Subfragments Muscle Smooth Cell Biology musculoskeletal system Actins Caldesmon chemistry Ionic strength Mutagenesis Site-Directed biology.protein Calmodulin-Binding Proteins Chickens Research Article Protein Binding |
| Zdroj: | Biochemical Journal. 328:211-218 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj3280211 |
| Popis: | Caldesmon interaction with smooth muscle myosin and its ability to cross-link actin filaments to myosin were investigated by the use of several bacterially expressed myosin-binding fragments of caldesmon. We have confirmed the presence of two functionally different myosin-binding sites located in domains 1 and 3/4a of caldesmon. The binding of the C-terminal site is highly sensitive to ionic strength and hardly participates in acto-myosin cross-linking, while the N-terminal binding site is relatively independent of ionic strength and apparently contains two separate myosin contact regions within residues 1-28 and 29-128 of chicken gizzard caldesmon. Both these N-terminal sub-sites are involved in the interaction with myosin and are predominantly responsible for the caldesmon-mediated high-affinity cross-linking of actin and myosin filaments, without affecting the affinity of direct acto-myosin interaction. Binding of caldesmon and its fragments to myosin or rod filaments revealed affinity in the micromolar range. We determined various stoichiometries at maximal binding, which depended on the ionic strength and the concentration of Mg2+ ions. At 30 mM NaCl and 1 mM Mg2+ the maximum stoichiometry was 4 moles of caldesmon (or caldesmon fragment) per mole of myosin. At 130 mM NaCl/1 mM Mg2+, or at 30 mM NaCl/5 mM Mg2+ it decreased to about two caldesmon molecules bound per myosin, while remaining 4:1 for individual caldesmon fragments, suggesting that all binding sequences on myosin were still fully capable of interaction. A further increase in the Mg2+ concentration led to a substantial decrease in both the affinity and maximum stoichiometry of caldesmon and the fragments binding to myosin. We suggest that caldesmon-myosin interaction varies according to the conformation of caldesmon in solution, that caldesmon-binding sites on myosin are not well defined and that their accessibility is determined by spatial organization and is blocked by divalent cations like Mg2+. |
| Databáze: | OpenAIRE |
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