LOCATION AND FUNCTIONAL-CHARACTERIZATION OF MYOSIN CONTACT SITES IN SMOOTH-MUSCLE CALDESMON

Caldesmon interaction with smooth muscle myosin and its ability to cro ss-link actin filaments to myosin were investigated by the use of seve ral bacterially expressed myosin-binding fragments of caldesmon. We ha ve 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 parti cipates in acto-myosin crosslinking, while the N-terminal binding site is relatively independent of ionic strength and apparently contains t wo separate myosin contact regions within residues 1-28 and 29-128 of chicken gizzard caldesmon. Both these N-terminal sub-sites are involve d in the interaction with myosin and are predominantly responsible for the caldesmon-mediated high-affinity cross-linking of actin and myosi n filaments, without affecting the affinity of direct acto-myosin inte raction. Binding of caldesmon and its fragments to myosin or rod filam ents revealed affinity in the micromolar range. We determined various stoichiometries at maximal binding, which depended on the ionic streng th 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, w hile remaining 4:1 for individual caldesmon fragments, suggesting that all binding sequences on myosin were still fully capable of interacti on. A further increase in the Mg2+ concentration led to a substantial decrease in both the affinity and maximum stoichiometry of caldesmon a nd the fragments binding to myosin. We suggest that caldesmon-myosin i nteraction varies according to the conformation of caldesmon in soluti on, that caldesmon-binding sites on myosin are not well defined and th at their accessibility is determined by spatial organization and is bl ocked by divalent cations like Mg2+.