Bio-X Center, Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, 2 Yikuang Street, Harbin 150080, China.
Int J Biochem Cell Biol. 2010 Jun;42(6):890-901. doi: 10.1016/j.biocel.2010.01.019. Epub 2010 Jan 21.
Trigger factor (TF) is the first chaperone encountered by nascent chains in bacteria, which consists of two modules: peptidyl-prolyl-cis/trans-isomerase (PPIase) domain and a crevice built by both N- and C-terminal domains. While the crevice is suggested to provide a protective space over the peptide exit site of ribosome for nascent polypeptides to fold, it remains unclear whether PPIase domain is directly involved in assisting protein folding. Here, we introduced structural change into different regions of TF, and investigated their influence on the chaperone function of TF in assisting the folding of various substrate proteins, including oligomeric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monomeric carbonic anhydrase II (CA II) and lysozyme. Results showed that structural disturbances by site-specific mutations in the PPIase active site or by deletion of the PPIase domain from TF affected the chaperone activity of TF toward CA II and GAPDH but had no effect on TF-assisted lysozyme refolding, suggesting PPIase domain is involved in assisting the folding of substrates larger than lysozyme. Mutants with the structural disturbances in the crevice totally lost the chaperone activity toward all the substrates we used in this investigation. These results provide further evidence to confirm that the crevice is the major chaperone site of TF, and the hydrophobic pocket in PPIase domain acts as an auxiliary site to assist the folding of substrate proteins bound to the crevice in a substrate-dependent manner, which is beneficial for TF to provide appropriate assistance for protein folding by changing protective space and binding affinity.
触发因子（TF）是细菌中新生肽链遇到的第一种伴侣蛋白，由两个模块组成：肽基脯氨酰顺/反异构酶（PPIase）结构域和由 N 端和 C 端结构域构成的裂缝。虽然裂缝被认为为新生多肽提供了一个在核糖体肽出口处的保护空间，使其能够折叠，但 PPIase 结构域是否直接参与协助蛋白质折叠仍不清楚。在这里，我们引入了 TF 不同区域的结构变化，并研究了它们对 TF 协助各种底物蛋白折叠的伴侣功能的影响，包括寡聚甘油醛-3-磷酸脱氢酶（GAPDH）和单体碳酸酐酶 II（CA II）和溶菌酶。结果表明，PPIase 活性位点的定点突变或从 TF 中删除 PPIase 结构域引起的结构干扰，影响了 TF 对 CA II 和 GAPDH 的伴侣活性，但对 TF 辅助溶菌酶重折叠没有影响，这表明 PPIase 结构域参与协助比溶菌酶更大的底物折叠。在裂缝处具有结构干扰的突变体完全丧失了对我们在这项研究中使用的所有底物的伴侣活性。这些结果进一步证实了裂缝是 TF 的主要伴侣位点，PPIase 结构域中的疏水口袋作为辅助位点，以底物依赖的方式协助结合在裂缝中的底物蛋白的折叠，这有利于 TF 通过改变保护空间和结合亲和力为蛋白质折叠提供适当的帮助。
