Helix A stabilization precedes amino-terminal lobe activation upon calcium binding to calmodulin
Helix A stabilization precedes amino-terminal lobe activation upon calcium binding to calmodulin
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DOI：
10.1021/bi800566u
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发表时间：
2008-09-02
期刊：
影响因子：
2.9
通讯作者：
Squier, Thomas C.
中科院分区：
文献类型：
--
作者：
Chen, Baowei;Lowry, David F.;Squier, Thomas C.
使用构象敏感的双砷探针4，5-双（1，3，2-二硫阿索烷-2-基）试卤灵（ReAsH）研究了CaM的相对结构域之间的结构偶联，所述双砷探针在与螺旋A（Thr-5至Phe-19）末端附近的工程化四半胱氨酸基序结合后变得高度荧光。构象和动力学的变化反映了天然CaM结构，因为与野生型CaM相比，（1）H-（15）N HSQC NMR谱没有变化。我们发现的证据与钙调素激活，其中钙占据的氨基末端和羧基末端叶钙调素差异影响绑定的ReAsH的荧光强度的网站的构象中间体。洞察的构象中间体的结构是可能的，从考虑钙依赖性的变化率的ReAsH结合和螺旋A的流动性，分别区分二级结构的变化与螺旋A的稳定性从三级结构重组的氨基末端叶钙调素必要的高亲和力结合到靶蛋白。受阻胺A的稳定性与羧基末端叶部位的钙占据有关（K（d）= 0.36 +/-0.04 μ M），这导致ReAsH结合速率从4900 M（-1）s（-1）降至370 M（-1）s（-1）。相比之下，涉及螺旋A和氨基末端叶中的其他结构元件的三级结构变化需要氨基末端位点的钙占据（K（d）= 18 +/-3 μ M）。所观察到的二级和三级结构的变化，涉及螺旋A的羧基和氨基末端叶钙结合位点的顺序钙占用表明一个重要的参与螺旋A介导的结构耦合之间的相对结构域的钙调素。这些结果进行了讨论，在一个模型中，羧基末端叶钙激活诱导二级结构的变化内的域间连接器，释放螺旋A，从而促进钙结合位点的形成在氨基末端叶和连接的三级结构重排，形成一个高亲和力的结合裂缝，可以与靶蛋白。
The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)resorufin (ReAsH), which upon binding to an engineered tetracysteine motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the (1)H-(15)N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH. Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe (K(d) = 0.36 +/- 0.04 mu M), which results in a reduction in the rate of ReAsH binding from 4900 M(-1) s(-1) to 370 M(-1) s(-1). In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe require calcium occupancy of amino-terminal sites (K(d) = 18 +/- 3 mu M). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces secondary structural changes within the interdomain linker that release helix A, thereby facilitating the formation of calcium binding sites in the amino-terminal lobe and linked tertiary structural rearrangements to form a high-affinity binding cleft that can associate with target proteins.