Target recognition by calmodulin: Dissecting the kinetics and affinity of interaction using short peptide sequences
Target recognition by calmodulin: Dissecting the kinetics and affinity of interaction using short peptide sequences
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DOI：
10.1002/pro.5560050701
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发表时间：
1996-07-01
期刊：
影响因子：
8
通讯作者：
Martin, SR
中科院分区：
文献类型：
--
作者：
Bayley, PM;Findlay, WA;Martin, SR
钙调蛋白（CaM）和肽M13（其来自骨骼肌肌球蛋白轻链激酶的靶结合序列）之间的相互作用主要涉及两组相互作用，即N-末端靶残基和钙调蛋白的C-结构域之间的相互作用，以及C-末端靶残基和钙调蛋白的N-结构域之间的相互作用（Ikura M等人，1992，Science 256：632-638）。使用短的合成肽的基础上的两个半的目标序列，与钙调素和其单独的C-结构域的相互作用进行了研究，通过荧光和CD光谱，钙结合，和动力学技术。肽WF 10（M13的残基1-10）与CaM结合，K-d约为1 μ M;肽FW 10（M13的残基9-18，具有Phe-17 -> Trp取代）与CaM结合，K-d约为100 μ M。肽WF 10对钙与钙调蛋白结合的影响产生双相饱和曲线，两个钙离子与C结构域结合的亲和力显著增强，形成稳定的半饱和复合物Ca-2-CaM-肽，并证实该序列与C结构域相互作用的功能重要性。停流研究表明，EGTA诱导的WF 10从Ca（4）CaM的解离是通过从动力学中间态的可逆弛豫机制进行的，也涉及CaM的半饱和，并且相同的机制对于完整的靶肽是明显的。N-末端靶残基与C-结构域的相互作用在能量上是最重要的组分，但是钙调蛋白与整个靶序列的相互作用对于诱导靶序列的两个连续元件与钙调蛋白的N-和C-结构域两者的完全协同相互作用是必要的。因此，钙调蛋白与M13序列的相互作用可以在结构和动力学基础上被分解为涉及包含靶序列的不同区域的中间体的部分反应。我们提出了一种钙调节钙调素依赖性酶激活的一般机制，涉及由钙调素C-结构域和靶序列的相应部分的相互作用形成的中间复合物。该中间物质可用于调节激活的总体钙敏感性并确定钙调蛋白靶相互作用的亲和力。
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 K-d approximate to 1 mu M; peptide FW10 (residues 9-18 of M13, with Phe-17 --> Trp substitution) binds to CaM with K-d approximate to 100 mu M. 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, Ca-2-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 Ca(4)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.