Ohki S, Ikura M, Zhang M
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon.
Biochemistry. 1997 Apr 8;36(14):4309-16. doi: 10.1021/bi962759m.
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.
使用二维核磁共振和荧光光谱技术研究了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，在没有Ca2+的情况下，Mg2+优先结合到CaM的Ca2+结合位点I和IV，并且Ca2+结合位点III对Mg2+的亲和力最低。与Ca2+结合引起的明显结构转变相反，Mg2+结合仅导致CaM的四个Ca2+结合环内的局部构象变化。因此，Mg2+似乎无法引起CaM与靶蛋白相互作用所需的显著结构效应。过量Mg2+（高达10 mM）的存在不会改变Ca2+与CaM结合的顺序和协同性，并且正如预期的那样，Ca2+饱和的CaM的结构不受Mg2+存在的影响。然而，我们发现Ca2+饱和的CaM与靶肽的结合受到Mg2+的影响，随着Mg2+浓度的增加，结合亲和力降低。研究了三种不同的肽，分别对应于骨骼肌肌球蛋白轻链激酶（MLCK）、CaM依赖性环核苷酸磷酸二酯酶（PDE）和平滑肌钙调蛋白（CaD）的CaM结合结构域，并显示它们对CaM的亲和力有不同程度的降低。在50 mM Mg2+存在下，MLCK肽对CaM的结合亲和力比在没有Mg2+时低约40倍，并且对PDE肽也观察到类似的反应。CaD肽对CaM的亲和力也显示出Mg2+依赖性，尽管程度要低得多。Mg2+依赖性地降低CaM与其靶肽之间的亲和力是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与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+浓度波动时在靶标激活中的特异性。
