Basic interdomain boundary residues in calmodulin decrease calcium affinity of sites I and II by stabilizing helix-helix interactions
Basic interdomain boundary residues in calmodulin decrease calcium affinity of sites I and II by stabilizing helix-helix interactions
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DOI：
10.1002/prot.10281
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发表时间：
2003-02-15
期刊：
影响因子：
--
通讯作者：
Shea, MA
中科院分区：
文献类型：
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作者：
Faga, LA;Sorensen, BR;Shea, MA
钙调素是一种EF-hand钙结合蛋白（148a.a），在细胞内信号转导中必不可少。它的同源N端和c端结构域被一个在核磁共振研究中表现为无序的连接体分开。在一项对草履虫CaM (PCaM1-75) n结构域片段的研究中，加入76 ~ 80 （MKEQD）连接体残基使T-m升高了9度，使钙结合降低了0.54 kcal/mol (Sorensen et al., Biochemistry 2002; 41:15-20)，表明这些系链残基影响能量学，同时也是扩散的屏障。为了确定残基74到80 （RKMKEQD）对稳定性和钙亲和力的单独贡献，我们比较了一系列嵌套的7个片段（PCaM1-74和PCaM1-80）。对于前4个PCaM1-74至PCaM1-77，在c端添加单个氨基酸对应于热稳定性的逐步增加和钙亲和力的降低，t的净变化为13.5℃，自由能为0.55 kcal/mol。PCaM1-77至PCaM1-80片段的热力学性质几乎相同。我们得出结论，从74到77 （RKMK）的3个碱基对较长的n域片段的稳定性增加和钙亲和力降低至关重要。N-15-PCaM1-74和N-15-PCaM1-80的核磁共振（HSQC）光谱比较和高分辨率结构模型分析表明，这些残基“锁定”在CaM的A螺旋氨基酸上。残基E78、Q79和D80的加入对位点I和II的影响很小，但它们可能有助于结构域之间的能量通讯机制。(C) 2003 Wiley-Liss, Inc。
Calmodulin is an EF-hand calcium-binding protein (148 a.a.) essential in intracellular signal transduction. Its homologous N- and C-terminal domains are separated by a linker that appears disordered in NMR studies. In a study of an N-domain fragment of Paramecium CaM (PCaM1-75), the addition of linker residues 76 to 80 (MKEQD) raised the T-m by 9degreesC and lowered calcium binding by 0.54 kcal/mol (Sorensen et al., Biochemistry 2002; 41:15-20), showing that these tether residues affect energetics as well as being a barrier to diffusion. To determine the individual contributions of residues 74 through 80 (RKMKEQD) to stability and calcium affinity, we compared a nested series of 7 fragments (PCaM1-74 to PCaM1-80). For the first 4, PCaM1-74 through PCaM1-77, single amino acid additions at the C-terminus corresponded to stepwise increases in thermostability and decreases in calcium affinity with a net change of 13.5degreesC in T. and 0.55 kcal/mol in free energy. The thermodynamic properties of fragments PCaM1-77 through PCaM1-80 were nearly identical. We concluded that the 3 basic residues in the sequence from 74 to 77 (RKMK) are critical to the increased stability and decreased calcium affinity of the longer N-domain fragments. Comparisons of NMR (HSQC) spectra of N-15-PCaM1-74 and N-15-PCaM1-80 and analysis of high-resolution structural models suggest these residues are "latched" to amino acids in helix A of CaM. The addition of residues E78, Q79, and D80 had a minimal effect on sites I and II, but they may contribute to the mechanism of energetic communication between the domains. (C) 2003 Wiley-Liss, Inc.