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Journal of Virology, September 2009, p. 9024-9030, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00911-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Nucleoside Monophosphate Complex Structures of the Endonuclease Domain from the Influenza Virus Polymerase PA Subunit Reveal the Substrate Binding Site inside the Catalytic Center {triangledown}

Cong Zhao,1,§ Zhiyong Lou,1,§ Yu Guo,2 Ming Ma,3 Yutao Chen,3 Shuaiyi Liang,1 Liang Zhang,2 Shoudeng Chen,3 Xuemei Li,3 Yingfang Liu,3* Mark Bartlam,2 and Zihe Rao1,2,3*

Structural Biology Laboratory, Tsinghua University, Beijing 100084, China,1 College of Life Sciences and Tianjin State Laboratory of Protein Science, Nankai University, Tianjin 300071, China,2 National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100101, China3

Received 7 May 2009/ Accepted 1 July 2009

Highly pathogenic influenza virus strains currently in circulation pose a significant risk of a global pandemic. Following the reported crystal structure of the endonuclease domain from the avian influenza virus polymerase PA subunit, here we report the results of a systematic X-ray crystallographic analysis of its complex with adenosine, uridine, and thymidine nucleoside monophosphates (NMPs). Electron density corresponding to the monophosphate moiety of each nucleotide was apparent in each NMP complex and bound to the catalytic metal. A hydrophobic site was found to contribute to nucleoside binding. The NMP complex structures should represent the conformation of the bound product after nuclease cleavage. Moreover, one solvent molecule was found to occupy an equivalent position to the second reported Mn2+ ion, where it mediates the interaction between bound NMPs and the N-terminal PA domain in the presence of the Mg2+ ion. The results presented here indicate a possible cleavage mechanism and identify a distinct nucleotide binding pocket. The identification of this binding pocket opens a new avenue for anti-influenza drug discovery, targeting the cap-dependent endonuclease, in response to the worldwide threat of influenza.

* Corresponding author. Mailing address for Zihe Rao: Laboratory of Structural Biology, Room 201, New Life Sciences Building, Tsinghua University, Beijing 100084, China. Phone: 86-10-62771493. Fax: 86-10-62773145. E-mail: raozh{at}xtal.tsinghua.edu.cn. Mailing address for Yingfang Liu: National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100101, China. Phone: 86-10-64888556. Fax: 86-10-64871293. E-mail: liuy{at}sun5.ibp.ac.cn

{triangledown} Published ahead of print on 8 July 2009.

§ These authors contributed equally to this work.

Journal of Virology, September 2009, p. 9024-9030, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00911-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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