Konno Michiko, Sano Yumi, Okudaira Kayoko, Kawaguchi Yoko, Yamagishi-Ohmori Yoko, Fushinobu Shinya, Matsuzawa Hiroshi
Department of Chemistry, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan.
Eur J Biochem. 2004 Sep;271(18):3794-803. doi: 10.1111/j.1432-1033.2004.04321.x.
Cyclophilins facilitate the peptidyl-prolyl isomerization of a trans-isomer to a cis-isomer in the refolding process of unfolded proteins to recover the natural folding state with cis-proline conformation. To date, only short peptides with a cis-form proline have been observed in complexes of human and Escherichia coli proteins of cyclophilin A, which is present in cytoplasm. The crystal structures analyzed in this study show two complexes in which peptides having a trans-form proline, i.e. succinyl-Ala-trans-Pro-Ala-p-nitroanilide and acetyl-Ala-Ala-trans-Pro-Ala-amidomethylcoumarin, are bound on a K163T mutant of Escherichia coli cyclophilin B, the preprotein of which has a signal sequence. Comparison with cis-form peptides bound to cyclophilin A reveals that in any case the proline ring is inserted into the hydrophobic pocket and a hydrogen bond between CO of Pro and Neta2 of Arg is formed to fix the peptide. On the other hand, in the cis-isomer, the formation of two hydrogen bonds of NH and CO of Ala preceding Pro with the protein fixes the peptide, whereas in the trans-isomer formation of a hydrogen bond between CO preceding Ala-Pro and His47 Nepsilon2 via a mediating water molecule allows the large distortion in the orientation of Ala of Ala-Pro. Although loss of double bond character of the amide bond of Ala-Pro is essential to the isomerization pathway occurring by rotating around its bond, these peptides have forms impossible to undergo proton transfer from the guanidyl group of Arg to the prolyl N atom, which induces loss of double bond character.
亲环蛋白在未折叠蛋白的重折叠过程中促进反式异构体向顺式异构体的肽基 - 脯氨酰异构化，以恢复具有顺式脯氨酸构象的天然折叠状态。迄今为止，在存在于细胞质中的亲环蛋白A的人和大肠杆菌蛋白复合物中，仅观察到具有顺式脯氨酸形式的短肽。本研究分析的晶体结构显示了两种复合物，其中具有反式脯氨酸的肽，即琥珀酰 - 丙氨酸 - 反式 - 脯氨酸 - 丙氨酸 - 对硝基苯胺和乙酰基 - 丙氨酸 - 丙氨酸 - 反式 - 脯氨酸 - 丙氨酸 - 氨基甲基香豆素，结合在大肠杆菌亲环蛋白B的K163T突变体上，其前体蛋白具有信号序列。与结合亲环蛋白A的顺式肽的比较表明，在任何情况下，脯氨酸环都插入疏水口袋中，并在Pro的CO和Arg的Neta2之间形成氢键以固定肽。另一方面，在顺式异构体中，Pro之前的Ala的NH和CO与蛋白质形成两个氢键来固定肽，而在反式异构体中，Ala - Pro的Ala之前的CO与His47 Nepsilon2之间通过介导水分子形成氢键，使得Ala - Pro的Ala取向发生大的扭曲。尽管Ala - Pro酰胺键双键性质的丧失对于通过围绕其键旋转发生的异构化途径至关重要，但这些肽具有不可能发生从Arg的胍基向脯氨酰N原子的质子转移的形式，这会导致双键性质的丧失。
