Chen Baowei, Lowry David F, Mayer M Uljana, Squier Thomas C
Cell Biology and Biochemistry Group, Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.
Biochemistry. 2008 Sep 2;47(35):9220-6. doi: 10.1021/bi800566u. Epub 2008 Aug 9.
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 microM), 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 microM). 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.
利用构象敏感的双砷探针4,5-双（1,3,2-二硫砷杂环戊烷-2-基）试卤灵（ReAsH）研究了钙调蛋白（CaM）相对结构域之间的结构偶联。该探针与A螺旋末端（苏氨酸-5至苯丙氨酸-19）附近的工程化四半胱氨酸基序结合后会发出高强度荧光。构象和动力学的变化反映了天然CaM的结构，因为与野生型CaM相比，（1）H-（15）N HSQC核磁共振谱没有变化。我们发现了与CaM激活相关的构象中间体的证据，其中CaM氨基末端和羧基末端叶中位点的钙占据差异影响结合的ReAsH的荧光强度。从ReAsH结合速率和A螺旋流动性的钙依赖性变化来考虑，有可能深入了解构象中间体的结构，这分别将与A螺旋稳定相关的二级结构变化与CaM氨基末端叶的三级结构重组区分开来，而后者是与靶蛋白高亲和力结合所必需的。A螺旋的稳定与羧基末端叶中位点的钙占据有关（Kd = 0.36 +/- 0.04 microM），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A螺旋和氨基末端叶中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（Kd = 18 +/- 3 microM）。观察到的响应羧基末端和氨基末端叶钙结合位点的顺序钙占据而涉及A螺旋的二级和三级结构变化表明，A螺旋在介导CaM相对结构域之间的结构偶联中起重要作用。这些结果将根据一个模型进行讨论，在该模型中，羧基末端叶钙激活诱导结构域间连接子内的二级结构变化，从而释放A螺旋，进而促进氨基末端叶中钙结合位点的形成以及相关的三级结构重排，以形成一个可与靶蛋白结合的高亲和力结合裂隙。
