Vorotnikov A V, Marston S B, Huber P A
Laboratory of Cell Motility, Institute of Experimental Cardiology, Russian Cardiology Research Centre, Moscow.
Biochem J. 1997 Nov 15;328 ( Pt 1)(Pt 1):211-8. doi: 10.1042/bj3280211.
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/5mM 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+.
利用几种细菌表达的钙调蛋白的肌球蛋白结合片段，研究了钙调蛋白与平滑肌肌球蛋白的相互作用及其将肌动蛋白丝交联到肌球蛋白的能力。我们已经证实，在钙调蛋白的结构域1和3/4a中存在两个功能不同的肌球蛋白结合位点。C末端位点的结合对离子强度高度敏感，几乎不参与肌动蛋白-肌球蛋白交联，而N末端结合位点相对独立于离子强度，并且在鸡胃钙调蛋白的1-28和29-128位残基内显然包含两个独立的肌球蛋白接触区域。这两个N末端亚位点都参与与肌球蛋白的相互作用，并且主要负责钙调蛋白介导的肌动蛋白和肌球蛋白丝的高亲和力交联，而不影响直接肌动蛋白-肌球蛋白相互作用的亲和力。钙调蛋白及其片段与肌球蛋白或杆状丝的结合显示出微摩尔范围内的亲和力。我们确定了最大结合时的各种化学计量比，这取决于离子强度和Mg2+离子的浓度。在30 mM NaCl和1 mM Mg2+条件下，最大化学计量比为每摩尔肌球蛋白4摩尔钙调蛋白（或钙调蛋白片段）。在130 mM NaCl/1 mM Mg2+或30 mM NaCl/5 mM Mg2+条件下，它降至每分子肌球蛋白约结合两个钙调蛋白分子，而单个钙调蛋白片段仍为4:1，这表明肌球蛋白上的所有结合序列仍完全能够相互作用。Mg2+浓度的进一步增加导致钙调蛋白及其片段与肌球蛋白结合的亲和力和最大化学计量比大幅下降。我们认为，钙调蛋白-肌球蛋白相互作用根据溶液中钙调蛋白的构象而变化，肌球蛋白上的钙调蛋白结合位点不明确，其可及性由空间组织决定，并被Mg2+等二价阳离子阻断。
