Change in Structure and Ligand Binding Properties of Hyperstable Cytochrome c555 from Aquifex aeolicus by Domain Swapping
Change in Structure and Ligand Binding Properties of Hyperstable Cytochrome c555 from Aquifex aeolicus by Domain Swapping
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通过域交换改变 Aquifex aeolicus 超稳定细胞色素 c555 的结构和配体结合特性
DOI：
10.1002/pro.2627
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发表时间：
2014
期刊：
影响因子：
8
通讯作者：
Y. Higuchi and S. Hirota
中科院分区：
文献类型：
--
作者：
M. Yamanaka;S. Nagao;H. Komori;Y. Higuchi and S. Hirota
Cytochromec 555（AA cytc 555）是嗜热菌Aquifex aeolicus的一种超稳定蛋白，属于cytc蛋白家族，具有独特的310-α-310长螺旋，含有血红素连接的Met 61。在此，我们发现AA cytc 555通过交换含有额外的310-α-310螺旋和C-末端α-螺旋的区域形成二聚体。二聚体AA cytc 555晶体的不对称单元包含两种二聚体结构，其中铰链区（Val 53-Lys 57）的结构在所有四种原聚体中是不同的。根据DSC测量，二聚体AA cytc 555在92 ± 1°C下解离成单体，表明二聚体是热稳定的。根据CD测量，二聚体AA cytc 555的二级结构保持在pH 2.2-11.0。CN-和CO分别以三价铁和亚铁态与二聚体AA cytc 555结合，这是由于二聚体中靠近Met 61的铰链区的柔性，而这些配体在相同条件下不与单体结合。此外，由于二聚体的热稳定性和pH耐受性，CN-和CO分别在中性pH和宽范围的pH（pH 2.2-11.0）、宽范围的温度（25-85°C）下与氧化和还原的二聚体结合。这些结果表明，超稳定的AA cytc 555的配体结合特性的变化后，通过结构域交换二聚体。
Cytochromec555from hyperthermophilic bacteriaAquifex aeolicus(AA cytc555) is a hyperstable protein belonging to the cytcprotein family, which possesses a unique long 310‐α‐310helix containing the heme‐ligating Met61. Herein, we show that AA cytc555forms dimers by swapping the region containing the extra 310‐α‐310helix and C‐terminal α‐helix. The asymmetric unit of the crystal of dimeric AA cytc555contained two dimer structures, where the structure of the hinge region (Val53–Lys57) was different among all four protomers. Dimeric AA cytc555dissociated to monomers at 92 ± 1°C according to DSC measurements, showing that the dimer was thermostable. According to CD measurements, the secondary structures of dimeric AA cytc555were maintained at pH 2.2–11.0. CN‐and CO bound to dimeric AA cytc555in the ferric and ferrous states, respectively, owing to the flexibility of the hinge region close to Met61 in the dimer, whereas these ligands did not bind to the monomer under the same conditions. In addition, CN‐and CO bound to the oxidized and reduced dimer at neutral pH and a wide range of pH (pH 2.2–11.0), respectively, in a wide range of temperature (25–85°C), owing to the thermostability and pH tolerance of the dimer. These results show that the ligand binding character of hyperstable AA cytc555changes upon dimerization by domain swapping.