Bayley P M, Findlay W A, Martin S R
Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London, United Kingdom.
Protein Sci. 1996 Jul;5(7):1215-28. doi: 10.1002/pro.5560050701.
The interaction between calmodulin (CaM) and peptide M13, its target binding sequence from skeletal muscle myosin light chain kinase, involves predominantly two sets of interactions, between the N-terminal target residues and the C-domain of calmodulin, and between the C-terminal target residues and the N-domain of calmodulin (Ikura M et al., 1992, Science 256:632-638). Using short synthetic peptides based on the two halves of the target sequence, the interactions with calmodulin and its separate C-domain have been studied by fluorescence and CD spectroscopy, calcium binding, and kinetic techniques. Peptide WF10 (residues 1-10 of M13) binds to CaM with Kd approximately 1 microM; peptide FW10 (residues 9-18 of M13, with Phe-17-->Trp substitution) binds to CaM with Kd approximately 100 microM. The effect of peptide WF10 on calcium binding to calmodulin produces a biphasic saturation curve, with marked enhancement of affinity for the binding of two calcium ions to the C-domain, forming a stable half-saturated complex, Ca2-CaM-peptide, and confirming the functional importance of the interaction of this sequence with the C-domain. Stopped-flow studies show that the EGTA-induced dissociation of WF10 from Ca4-CaM proceeds by a reversible relaxation mechanism from a kinetic intermediate state, also involving half-saturation of CaM, and the same mechanism is evident for the full target peptide. Interaction of the N-terminal target residues with the C-domain is energetically the most important component, but interaction of calmodulin with the whole target sequence is necessary to induce the full cooperative interaction of the two contiguous elements of the target sequence with both N- and C-domains of calmodulin. Thus, the interaction of calmodulin with the M13 sequence can be dissected on both a structural and kinetic basis into partial reactions involving intermediates comprising distinct regions of the target sequence. We propose a general mechanism for the calcium regulation of calmodulin-dependent enzyme activation, involving an intermediate complex formed by interaction of the calmodulin C-domain and the corresponding part of the target sequence. This intermediate species can function to regulate the overall calcium sensitivity of activation and to determine the affinity of the calmodulin target interaction.
钙调蛋白（CaM）与肽M13（其来自骨骼肌肌球蛋白轻链激酶的靶标结合序列）之间的相互作用主要涉及两组相互作用，即N端靶标残基与钙调蛋白C结构域之间的相互作用，以及C端靶标残基与钙调蛋白N结构域之间的相互作用（仓田真人等人，1992年，《科学》256:632 - 638）。使用基于靶标序列两半部分的短合成肽，通过荧光和圆二色光谱、钙结合及动力学技术研究了它们与钙调蛋白及其单独的C结构域的相互作用。肽WF10（M13的第1 - 10位残基）以约1微摩尔的解离常数（Kd）与CaM结合；肽FW10（M13的第9 - 18位残基，苯丙氨酸 - 17被色氨酸取代）以约100微摩尔的Kd与CaM结合。肽WF10对钙与钙调蛋白结合的影响产生双相饱和曲线，显著增强了两个钙离子与C结构域结合的亲和力，形成稳定的半饱和复合物Ca2 - CaM - 肽，证实了该序列与C结构域相互作用的功能重要性。停流研究表明，乙二醇双四乙酸（EGTA）诱导的WF10从Ca4 - CaM上解离是通过动力学中间态的可逆弛豫机制进行的，该中间态也涉及CaM的半饱和状态，并且完整的靶标肽也有相同的机制。N端靶标残基与C结构域的相互作用在能量上是最重要的组成部分，但钙调蛋白与整个靶标序列的相互作用对于诱导靶标序列的两个相邻元件与钙调蛋白的N结构域和C结构域的完全协同相互作用是必要的。因此，钙调蛋白与M13序列的相互作用在结构和动力学基础上都可以分解为涉及由靶标序列不同区域组成的中间体的部分反应。我们提出了一种钙调蛋白依赖性酶激活的钙调节的一般机制，涉及由钙调蛋白C结构域与靶标序列相应部分相互作用形成的中间复合物。这种中间物种可以起到调节激活的整体钙敏感性并确定钙调蛋白靶标相互作用亲和力的作用。
