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首页> 外文期刊>Biochemistry >Identification of Mg2+-binding sites and the role of Mg2+ on target recognition by calmodulin.
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Identification of Mg2+-binding sites and the role of Mg2+ on target recognition by calmodulin.

机译：Mg2 +结合位点的鉴定和钙调蛋白对目标识别中Mg2 +的作用。

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The binding of Mg2+ to calmodulin (CaM) and the effect of Mg2+ on the binding of Ca2+-CaM to target peptides were examined using two-dimensional nuclear magnetic resonance and fluorescence spectroscopic techniques. We found that Mg2+ preferentially binds to Ca2+-binding sites I and IV of CaM in the absence of Ca2+ and that Ca2+-binding site III displays the lowest affinity for Mg2+. In contrast to the marked structural transitions induced by Ca2+ binding, Mg2+ binding causes only localized conformational changes within the four Ca2+-binding loops of CaM. Therefore, Mg2+ does not seem to be able to cause significant structural effects required for the interaction of CaM with target proteins. The presence of excess Mg2+ (up to 10 mM) does not change the order and cooperativity of Ca2+ binding to CaM, and as expected, the structure of Ca2+-saturated CaM is not affected by the presence of Mg2+. However, we found that the binding of Ca2+-saturated CaM to target peptides is affected by Mg2+ with the binding affinity decreasing as the Mg2+ concentration increases. Three different peptides, corresponding to the CaM binding domain of skeletal muscle myosin light-chain kinase (MLCK), CaM-dependent cyclic nucleotide phosphodiesterase (PDE), and smooth muscle caldesmon (CaD), were examined and show different reductions in their affinities toward CaM. The CaM-binding affinity of the MLCK peptide in the presence of 50 mM Mg2+ is approximately 40-fold lower than that seen in the absence of Mg2+, and a similar response was observed for the PDE peptide. The affinity of the CaD peptide for CaM also shows a Mg2+ dependence, though to a much lower magnitude. The Mg2+-dependent decrease in the affinities between CaM and its target peptides is an intrinsic property of Mg2+ rather than a nonspecific ionic effect, as other metal ions such as Na+ do not completely replicate the effect of Mg2+. The inhibitory effect of Mg2+ on the formation of complexes between CaM and its targets may contribute to the specificity of CaM in target activation in response to cellular Ca2+ concentration fluctuations.
机译：使用二维核磁共振和荧光光谱技术检查了Mg2 +与钙调蛋白（CaM）的结合以及Mg2 +对Ca2 + -CaM与靶肽结合的影响。我们发现，在不存在Ca2 +的情况下，Mg2 +优先与CaM的Ca2 +结合位点I和IV结合，并且Ca2 +结合位点III对Mg2 +的亲和力最低。与Ca2 +结合诱导的明显结构转变相反，Mg2 +结合仅引起CaM的四个Ca2 +结合环内的局部构象变化。因此，Mg2 +似乎不能引起CaM与靶蛋白相互作用所需的显着结构效应。过量的Mg2 +（最多10mM）的存在不会改变Ca2 +与CaM结合的顺序和协同作用，并且正如所预期的那样，饱和的Ca2 +的CaM的结构不受Mg2 +的存在的影响。但是，我们发现Ca2 +饱和CaM与目标肽的结合受Mg2 +影响，并且随着Mg2 +浓度的增加，结合亲和力降低。检查了三种不同的肽，分别对应于骨骼肌肌球蛋白轻链激酶（MLCK）的CaM结合结构域，依赖于CaM的环状核苷酸磷酸二酯酶（PDE）和平滑肌caldesmon（CaD），并显示出它们对血友病亲和力的不同降低CaM。在存在50 mM Mg2 +的情况下，MLCK肽的CaM结合亲和力比在没有Mg2 +的情况下所见的CaM结合亲和力低约40倍，并且对于PDE肽也观察到类似的响应。 CaD肽对CaM的亲和力也显示出Mg2 +依赖性，尽管幅度要低得多。 CaM及其靶肽之间亲和力的Mg2 +依赖性降低是Mg2 +的固有特性，而不是非特异性离子效应，因为其他金属离子（如Na +）不能完全复制Mg2 +的效应。 Mg2 +对CaM及其靶标之间复合物形成的抑制作用可能有助于CaM在响应细胞Ca2 +浓度波动的靶标激活中的特异性。

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