Solution Structure of Clostridial Collagenase H and Its Calcium-Dependent Global Conformation Change
Solution Structure of Clostridial Collagenase H and Its Calcium-Dependent Global Conformation Change
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DOI：
10.1016/j.bpj.2013.02.022
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发表时间：
2013-04-02
影响因子：
3.4
通讯作者：
Murayama, Kazutaka
中科院分区：
文献类型：
--
作者：
Ohbayashi, Naomi;Matsumoto, Takashi;Murayama, Kazutaka
来自溶组织梭菌的胶原酶H（ColH）是由胶原酶模块（激活剂和肽酶结构域）、两个多囊肾病样结构域和一个胶原结合结构域组成的多模块蛋白。结构域间的构象及其变化对于理解ColH的功能具有重要意义。在这项研究中，小角X射线散射和有限的蛋白水解，揭示了在溶液中的ColH的域间排列。从头算珠模型表明，ColH采用锥形膨胀头的形状。在钙螯合条件下（与EGTA），整体结构进一步延长。刚体模型表明胶原酶模块在溶液中优选为封闭形式。有限的蛋白水解表明，蛋白酶的敏感性的ColH显着增加的钙螯合条件下，消化主要发生在域连接器区域。用荧光染料的荧光测量在分离后用有限的蛋白水解产物进行。结果表明，有限的蛋白水解产物表现出类似于全长ColH的荧光。这些发现表明，全长ColH在溶液中的构象是细长的形式，这种形式是钙依赖性地保持在结构域连接区。
Collagenase H (ColH) from Clostridium histolyticum is a multimodular protein composed of a collagenase module (activator and peptidase domains), two polycystic kidney disease-like domains, and a collagen-binding domain. The interdomain conformation and its changes are very important for understanding the functions of ColH. In this study, small angle x-ray scattering and limited proteolysis were employed to reveal the interdomain arrangement of ColH in solution. The ab initio beads model indicated that ColH adopted a tapered shape with a swollen head. Under calcium-chelated conditions (with EGTA), the overall structure was further elongated. The rigid body model indicated that the closed form of the collagenase module was preferred in solution. The limited proteolysis demonstrated that the protease sensitivity of ColH was significantly increased under the calcium-chelated conditions, and that the digestion mainly occurred in the domain linker regions. Fluorescence measurements with a fluorescent dye were performed with the limited proteolysis products after separation. The results indicated that the limited proteolysis products exhibited fluorescence similar to that of the full-length ColH. These findings suggested that the conformation of full-length ColH in solution is the elongated form, and this form is calcium-dependently maintained at the domain linker regions.