Nucleoside Monophosphate Complex Structures of the Endonuclease Domain from the Influenza Virus Polymerase PA Subunit Reveal the Substrate Binding Site inside the Catalytic Center |
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| Authors: | Cong Zhao Zhiyong Lou Yu Guo Ming Ma Yutao Chen Shuaiyi Liang Liang Zhang Shoudeng Chen Xuemei Li Yingfang Liu Mark Bartlam Zihe Rao |
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| Affiliation: | 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. |
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| Abstract: | 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.The recent emergence of highly pathogenic avian and swine influenza viruses poses a significant global threat to human health (8). A total of 421 human infections by avian influenza H5N1 viruses have been reported worldwide since 2003, with 257 fatalities (WHO, April 2009). The current global outbreak of swine influenza with the H1N1 subtype has resulted in more than 100 deaths since it emerged in March 2009 and has spread to almost 40 countries. While approved anti-influenza drugs are available, their effectiveness in the event of an influenza pandemic may be limited due to drug resistance of the influenza viruses. Elucidating the underlying mechanisms of the virus life cycle and identifying new targets to be exploited for the discovery of antiviral therapeutics are therefore paramount.The influenza virus contains a segmented RNA genome with eight negative-sense segments encoding 11 proteins. The influenza virus polymerase is a heterotrimeric ∼250-kDa complex with the following three protein subunits: PA, PB1, and PB2. It plays central roles in the viral life cycle and is directly responsible for RNA synthesis for both viral replication and transcription. However, the mechanisms by which these two different RNA synthesis functions are regulated within the large polymerase complex remain unclear. There is still some controversy about the functions of the various subunits, which have been reviewed by Liu et al. previously (17). Briefly, PB1 contains conserved and well-characterized RNA-dependent RNA polymerase motifs (3), while PB2 is required for transcription (16) and methylated cap binding (7, 9). PA has been implicated in a diverse range of functions but has been confirmed to possess endonuclease activity (6, 23).PA is a 80-kDa subunit and can be cleaved into two independent domains (10, 11), as follows: a smaller N-terminal domain with cap-dependent endonuclease activity (6, 23) and a larger C-terminal domain that mediates the interaction with PB1 (13, 20). The recent crystal structures of the N-terminal PA domain, termed PAN, confirmed its endonuclease activity (6, 23), although the mode of substrate binding, cleavage mechanism, and metal dependence by PAN remains unclear. Understanding such aspects should provide a handle for the discovery of specific drugs that block the cap-snatching step during influenza virus genome replication. To provide a structural basis for substrate binding by the cap-dependent endonuclease, we have determined the high-resolution crystal structures of complexes of PAN with three nucleoside monophosphates (NMPs). For these three NMP complexes, ribo-UMP (rUMP), rAMP, and TMP, we observe electron density near to the active site which can be readily interpreted as the phosphate moieties of the NMPs. An additional solvent molecule occupies a potential metal ion binding site and mediates the interaction between the phosphate moieties of the NMPs and residues Glu119 and Lys134 of PAN. Moreover, the location of their less-ordered nucleoside moieties indicates a relatively hydrophobic pocket (N site). His41 is also involved in both the N site and the ribose binding site (R site). In sharp contrast, unambiguous electron density indicates that rGMP and rCMP will not bind into this site, suggesting that this binding site has substrate specificity. The results reported here provide a more-detailed picture of PAN nuclease cleavage and provide a distinct binding pocket for anti-influenza drug discovery targeting the cap-dependent endonuclease. |
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