Competition between intradomain and interdomain interactions: a buried salt bridge is essential for villin headpiece folding and actin binding.
Competition between intradomain and interdomain interactions: a buried salt bridge is essential for villin headpiece folding and actin binding.
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DOI：
10.1021/bi1020343
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发表时间：
2011-05-10
期刊：
影响因子：
2.9
通讯作者：
McKnight CJ
中科院分区：
文献类型：
--
作者：
Packer LE;Song B;Raleigh DP;McKnight CJ
Villin型头片段结构域是位于多种肌动蛋白相关蛋白C-末端的~70个残基序。Villin headpiece（HP 67）是蛋白质折叠研究中常用的模型系统。HP 67由两个子域组成，它们形成紧密堆积的界面。HP 67（HP 35）的C-末端亚结构域是已知的最小的折叠蛋白质之一。N-末端亚结构域需要C-末端亚结构域的存在才能折叠。在HP 67的结构中，保守的盐桥连接N-和C-末端亚结构域。残基E39和K70之间的这种埋藏盐桥在小蛋白质结构域中是不寻常的。我们使用突变分析，监测CD和NMR，和功能测定，以确定这个埋藏的盐桥的作用。首先，将盐桥中的两个残基用严格疏水的氨基酸E39 M/K70 M替换。第二，盐桥中的两个残基交换，E39 K/K70 E。与野生型盐桥残基的任何变化导致N-末端亚结构域的解折叠，即使当突变在HP 67的稳定变体中进行时。C-末端亚结构域在所有突变体中保持折叠，并且通过一些突变而稳定。使用肌动蛋白沉降分析，我们发现，一个折叠的N-末端结构域是必不可少的特异性肌动蛋白结合。因此，掩埋的盐桥是必需的特定折叠的N-末端结构域赋予肌动蛋白结合活性绒毛型头片段域，即使这种特定的相互作用所需的残基不稳定的C-末端亚结构域。
Villin-type headpiece domains are ~70 residue motifs that reside at the C-terminus of a variety of actin-associated proteins. Villin headpiece (HP67) is a commonly used model system for both experimental and computational studies of protein folding. HP67 is made up of two subdomains that form a tightly packed interface. The isolated C-terminal subdomain of HP67 (HP35) is one of the smallest autonomously-folding proteins known. The N-terminal subdomain requires the presence of the C-terminal subdomain to fold. In the structure of HP67, a conserved salt bridge connects N- and C-terminal subdomains. This buried salt bridge between residues E39 and K70 is unusual in a small protein domain. We used mutational analysis, monitored by CD and NMR, and functional assays to determine the role of this buried salt bridge. First, the two residues in the salt bridge were replaced with strictly hydrophobic amino acids, E39M/K70M. Second, the two residues in the salt bridge were swapped, E39K/K70E. Any change from the wild-type salt bridge residues results in unfolding of the N-terminal subdomain, even when the mutations were made in stabilized variant of HP67. The C-terminal subdomain remains folded in all mutants and is stabilized by some of the mutations. Using actin sedimentation assays we find that a folded N-terminal domain is essential for specific actin binding. Therefore, the buried salt bridge is required for the specific folding of the N-terminal domain which confers actin-binding activity to villin-type headpiece domains, even though the residues required for this specific interaction destabilize the C-terminal subdomain.
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影响因子：
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