Chaperone-Like Effect of the Linker on the Isolated C-Terminal Domain of Rabbit Muscle Creatine Kinase
Chaperone-Like Effect of the Linker on the Isolated C-Terminal Domain of Rabbit Muscle Creatine Kinase
复制标题
连接子对兔肌肌酸激酶分离 C 端结构域的分子伴侣样作用
DOI：
10.1016/j.bpj.2012.07.002
复制
发表时间：
2012-08-08
影响因子：
3.4
通讯作者：
Yan, Yong-Bin
中科院分区：
文献类型：
--
作者：
Chen, Zhe;Chen, Xiang-Jun;Yan, Yong-Bin
分子内分子伴侣（intramolecular chaperones，IMCs）是蛋白质一级结构中编码的特异性结构域/片段，具有分子伴侣样活性，可抑制同一分子中其他结构域的聚集。在本研究中，我们发现接头的截短大大促进了兔肌肌酸激酶（RMCK）C端结构域（CTD）的热聚集。共价连接到CTD的接头的存在或合成接头肽的供应另外可以以浓度依赖性方式成功地保护RMCK的CTD免于聚集。接头的截短片段也表现为具有较低效率的伴侣样效应，揭示了其C-末端一半在接头的IMC功能中的重要性。通过分子动力学模拟确定了RMCK CTD中的聚集位点。三个关键疏水性残基的突变分析导致对CTD与完整或部分接头之间的热聚集的相反影响，证实了接头作为盖子以保护疏水性残基免于暴露于溶剂的作用。这些观察结果表明，多结构域蛋白中的接头可以充当IMC，以促进聚集倾向结构域的正确折叠。此外，蛋白水解后IMC接头的完整性调节了非途径聚集体的产生，这对于由聚集的蛋白水解片段的毒性作用引起的一些疾病的发作可能是重要的。
Intramolecular chaperones (IMCs), which are specific domains/segments encoded in the primary structure of proteins, exhibit chaperone-like activity against the aggregation of the other domains in the same molecule. In this research, we found that the truncation of the linker greatly promoted the thermal aggregation of the isolated C-terminal domain (CTD) of rabbit muscle creatine kinase (RMCK). Either the existence of the linker covalently linked to CTD or the supply of the synthetic linker peptide additionally could successfully protect the CTD of RMCK against aggregation in a concentration-dependent manner. Truncated fragments of the linker also behaved as a chaperone-like effect with lower efficiency, revealing the importance of its C-terminal half in the IMC function of the linker. The aggregation sites in the CTD of RMCK were identified by molecular dynamics simulations. Mutational analysis of the three key hydrophobic residues resulted in opposing effects on the thermal aggregation between the CTD with intact or partial linker, confirming the role of linker as a lid to protect the hydrophobic residues against exposure to solvent. These observations suggested that the linkers in multidomain proteins could act as IMCs to facilitate the correct folding of the aggregation-prone domains. Furthermore, the intactness of the IMC linker after proteolysis modulates the production of off-pathway aggregates, which may be important to the onset of some diseases caused by the toxic effects of aggregated proteolytic fragments.