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首页> 外文期刊>Biochemistry >Low affinity Ca2+-binding sites of calcineurin B mediate conformational changes in calcineurin A.
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Low affinity Ca2+-binding sites of calcineurin B mediate conformational changes in calcineurin A.

机译：钙调神经磷酸酶B的低亲和力Ca2 +结合位点介导钙调神经磷酸酶A的构象变化。

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摘要

Limited proteolysis of calcineurin in the presence of Ca(2+) suggested that its calmodulin-binding domain, readily degraded by proteases, was unfolded while calcineurin B was compactly folded [Hubbard, M. J., and Klee, C. B. (1989) Biochemistry 28, 1868-1874]. Moreover, in the crystal structure of calcineurin, with the four Ca(2+) sites of calcineurin B occupied, the calmodulin-binding domain is not visible in the electron density map [Kissinger, C. R., et al. (1995) Nature 378, 641-644]. Limited proteolysis of calcineurin in the presence of EGTA, shows that, when the low affinity sites of calcineurin B are not occupied, the calmodulin-binding domain is completely protected against proteolytic attack. Slow cleavages are, however, detected in the linker region between the calmodulin-binding and the autoinhibitory domains of calcineurin A. Upon prolonged exposure to the protease, selective cleavages in carboxyl-terminal end of the first helix and the central helix linker of calcineurin B and the calcineurin B-binding helix of calcineurin A are also detected. Thus, Ca(2+) binding to the low-affinity sites of calcineurin B affects the conformation of calcineurin B and induces a conformational change of the regulatory domain of calcineurin A, resulting in the exposure of the calmodulin-binding domain. This conformational change is needed for the partial activation of the enzyme in the absence of calmodulin and its full activation by calmodulin. A synthetic peptide corresponding to the calmodulin-binding domain is shown to interact with a peptide corresponding to the calcineurin B-binding domain, and this interaction is prevented by calcineurin B in the presence but not the absence of Ca(2+). These observations provide a mechanism to explain the dependence on Ca(2+) binding to calcineurin B for calmodulin activation and for the 10-20-fold increase in affinity of calcineurin for Ca(2+) upon removal of the regulatory domain by limited proteolysis [Stemmer, P. M., and Klee, C. B. (1994) Biochemistry 33, 6859-6866].
机译：钙调神经磷酸酶在Ca（2+）存在下的有限蛋白水解表明其钙调蛋白结合结构域很容易被蛋白酶降解，而钙调神经磷酸酶B紧密折叠而未折叠[Hubbard，MJ，and Klee，CB（1989）Biochemistry 28，1868 -1874]。此外，在钙调神经磷酸酶的晶体结构中，钙调神经磷酸酶B的四个Ca（2+）位点被占据，钙调蛋白结合结构域在电子密度图中不可见[Kissinger，C.R。等人。 （1995）Nature 378，641-644]。在存在EGTA的情况下，钙调神经磷酸酶的蛋白水解作用有限，这表明，当钙调神经磷酸酶B的低亲和力位点不被占据时，钙调蛋白结合域就可以免受蛋白水解攻击。但是，在钙调蛋白结合和钙调神经磷酸酶A的自抑制域之间的接头区域中检测到慢速裂解。长时间暴露于蛋白酶后，第一个螺旋的羧基末端和钙调神经磷酸酶B的中央螺旋接头选择性裂解并且还检测到钙调神经磷酸酶A的钙调神经磷酸酶B结合螺旋。因此，Ca（2+）结合钙调神经磷酸酶B的低亲和力站点影响钙调神经磷酸酶B的构象，并诱导钙调神经磷酸酶A调节域的构象变化，从而导致钙调蛋白结合域的暴露。在不存在钙调蛋白的情况下，部分构象是酶的部分活化所必需的，而在钙调蛋白的完全活化下，则需要这种构象变化。对应于钙调蛋白结合结构域的合成肽显示与对应于钙调神经磷酸酶B结合结构域的肽相互作用，并且该相互作用在存在但不存在Ca（2+）的情况下被钙调神经磷酸酶B阻止。这些观察结果提供了一种机制，以解释对钙（钙调神经磷酸酶）Ca（2+）结合的依赖性，从而促进钙调蛋白的活化以及钙调神经磷酸酶对钙（2+）的亲和力通过有限的蛋白水解作用去除调控域后增加10-20倍[Stemmer，PM和Klee，CB（1994）Biochemistry 33，6859-6866]。

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