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Crystal structure of an archaeal Ski2p-like protein from Pyrococcus horikoshii OT3

¹ Laboratory of Structural Biology, Graduate School of Systems Life Sciences, Kyushu University, Fukuoka 812-8581, Japan
² Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Graduate School, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
³ Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan

(RECEIVED July 5, 2007; FINAL REVISION September 24, 2007; ACCEPTED September 27, 2007)

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 -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.

Keywords: archaea; DExD/H-box protein; HhH-fold; mRNA degradation; P. horikoshii ; RNA helicase; Ski2p; Ski complex; WH-fold; X-ray crystallography

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.073107008.

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