Dimer-oligomer interconversion of wild-type and mutant rat 2-Cys peroxiredoxin
Dimer-oligomer interconversion of wild-type and mutant rat 2-Cys peroxiredoxin
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DOI：
10.1074/jbc.m705753200
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发表时间：
2008-01-04
影响因子：
4.8
通讯作者：
Abe, Yasuko
中科院分区：
文献类型：
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作者：
Matsumura, Tomohiro;Okamoto, Ken;Abe, Yasuko
大鼠血红素结合蛋白 23 (HBP23)/过氧化还原蛋白 (Prx I) 属于 2-Cys 过氧化还原蛋白 I 型家族，具有与还原硫氧还蛋白 (Trx) 结合作为电子供体的过氧化物酶活性。我们通过凝胶过滤分析了野生型和突变体HBP23/Prx I的二聚体-寡聚体相互转化，发现C52S和C173S突变体主要以十聚体形式存在，而野生型是各种形式的混合物，在较高的蛋白质浓度和较低的离子盐浓度以及二硫苏糖醇存在下有利于十聚体。 C83S突变体主要是二聚体，与之前的晶体学分析一致(Hirotsu, S.、Abe, Y.、Okada, K.、Nagahara, N.、Hori, H.、Nishino, T.和Hakoshima, T.(1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12333-12338)。十聚C52S突变体的X射线衍射分析揭示了环形结构（直径，类似于130埃；内径，类似于55埃；厚度，类似于45埃）。与最近报道的人 Prx I 主要以在所有二聚体-二聚体界面处具有 Cys(83)-Cys(83) 二硫键的十聚体形式存在相反，大鼠 HBP23/Prx I 仅在一个二聚体-二聚体界面处具有 Cys(83)-Cys(83) 二硫键（S-S 分离类似于 2.1 埃），而其他界面处的相互作用（平均 S-S） 3.6 埃的分离）似乎涉及疏水力和范德华力。这一发现与凝胶过滤分析一致，表明该蛋白质很容易在二聚体和寡聚体形式之间相互转化。 C83S突变体在Trx/Trx还原酶系统中表现出与野生型相似的过氧化物酶活性，野生型完全是二聚体。在浓度较高时，蛋白质主要为十聚体，在 [C-14] 碘乙酰胺掺入实验中观察到还原 Trx 的攻击效率较低。我们认为二聚体-十聚体相互转化可能具有调节作用。
Rat heme-binding protein 23 (HBP23)/peroxiredoxin (Prx I) belongs to the 2-Cys peroxiredoxin type I family and exhibits peroxidase activity coupled with reduced thioredoxin (Trx) as an electron donor. We analyzed the dimer-oligomer interconversion of wild-type and mutant HBP23/Prx I by gel filtration and found that the C52S and C173S mutants existed mostly as decamers, whereas the wild type was a mixture of various forms, favoring the decamer at higher protein concentration and lower ionic salt concentration and in the presence of dithiothreitol. The C83S mutant was predominantly dimeric, in agreement with a previous crystallographic analysis (Hirotsu, S., Abe, Y., Okada, K., Nagahara, N., Hori, H., Nishino, T., and Hakoshima, T.(1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12333-12338). X-ray diffraction analysis of the decameric C52S mutant revealed a toroidal structure (diameter, similar to 130 angstrom; inside diameter, similar to 55 angstrom; thickness, similar to 45 angstrom). In contrast to human Prx I, which was recently reported to exist predominantly as the decamer with Cys(83)-Cys(83) disulfide bonds at all dimer-dimer interfaces, rat HBP23/Prx I has a Cys(83)-Cys(83) disulfide bond at only one dimer-dimer interface (S-S separation of similar to 2.1 angstrom), whereas the interactions at the other interfaces (mean S-S separation of 3.6 angstrom) appear to involve hydrophobic and van der Waals forces. This finding is consistent with gel filtration analyses showing that the protein readily interconverts between dimer and oligomeric forms. The C83S mutant exhibited similar peroxidase activity to the wild type, which is exclusively dimeric, in the Trx/Trx reductase system. Athigher concentrations, where the protein was mostly decameric, less efficient attack of reduced Trx was observed in a [C-14] iodoacetamide incorporation experiment. We suggest that the dimer-decamer interconversion may have a regulatory role.