Crystal structure of an archaeal Ski2p‐like protein from Pyrococcus horikoshii OT3
Crystal structure of an archaeal Ski2p‐like protein from Pyrococcus horikoshii OT3
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DOI：
10.1110/ps.073107008
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发表时间：
2007-11
期刊：
影响因子：
8
通讯作者：
Xiaodong Zhang;T. Nakashima;Y. Kakuta;M. Yao;I. Tanaka;M. Kimura
中科院分区：
文献类型：
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作者：
Xiaodong Zhang;T. Nakashima;Y. Kakuta;M. Yao;I. Tanaka;M. Kimura
由Ski 2 p、Ski 3 p和Ski 8 p组成的Ski复合物在酵母3′端至5′端胞质mRNA降解途径中起重要作用。Ski 2 p是一种假定的RNA解旋酶，属于DExD/H-box蛋白家族，在真核生物和古细菌中保守。来自超嗜热古菌Pyrococcus horikoshii OT 3的基因产物（Ph 1280 p）显示与Ski 2 p的序列同源性，与Ski 2 p的中心区域共享22.6%相同的氨基酸。为了获得Ski 2 p-样RNA解旋酶的结构信息，我们在大肠杆菌细胞中过量生产Ph 1280 p，并将其纯化至表观同质性。Ph 1280 p具有DNA/RNA依赖的ATP酶活性，最适温度为1290 °C。Ph 1280 p的晶体结构已经使用单波长反常色散（SAD）和硒代甲硫酰（Se-Met）取代蛋白质以3.5 μ m的分辨率解析。Ph 1280 p包含四个亚结构域;两个N末端亚结构域（N1和N2）折叠成具有保守解旋酶基序的RecA样结构，而两个C末端亚结构域（C1和C2）折叠成分别含有翼状螺旋（WH）折叠和螺旋-发夹-螺旋（HhH）折叠的α螺旋结构。虽然每个Ph 1280 p亚结构域的结构可以单独叠加在其他解旋酶（如大肠杆菌DNA解旋酶RecQ）中的相应结构域上，但Ph 1280 p中解旋酶和C末端亚结构域的相对取向与其他解旋酶显著不同。这种结构特征与Ski 2样解旋酶的底物特异性有关，并在Ski复合物中3′至5′细胞质mRNA降解中发挥关键作用。
The Ski complex composed of Ski2p, Ski3p, and Ski8p plays an essential role in the 3′ to 5′ cytoplasmic mRNA degradation pathway in yeast. Ski2p is a putative RNA helicase, belonging in the DExD/H‐box protein families and conserved in eukarya as well as in archaea. The gene product (Ph1280p) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 shows sequence homology with Ski2p, sharing 22.6% identical amino acids with a central region of Ski2p. In order to gain structural information about the Ski2p‐like RNA helicase, we overproduced Ph1280p in Escherichia coli cells, and purified it to apparent homogeneity. Ph1280p exhibits DNA/RNA‐dependent ATPase activity with an optimal temperature at ∼90°C. The crystal structure of Ph1280p has been solved at a resolution of 3.5 Å using single‐wavelength anomalous dispersion (SAD) and selenomethionyl (Se‐Met)‐substituted protein. Ph1280p comprises four subdomains; the two N‐terminal subdomains (N1 and N2) fold into an RecA‐like architecture with the conserved helicase motifs, while the two C‐terminal subdomains (C1 and C2) fold into α‐helical structures containing a winged helix (WH)‐fold and helix‐hairpin‐helix (HhH)‐fold, respectively. Although the structure of each of the Ph1280p subdomains can be individually superimposed on the corresponding domains in other helicases, such as the Escherichia coli DNA helicase RecQ, the relative orientation of the helicase and C‐terminal subdomains in Ph1280p is significantly different from that of other helicases. This structural feature is implicated in substrate specificity for the Ski2‐like helicase and would play a critical role in the 3′ to 5′ cytoplasmic mRNA degradation in the Ski complex.