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Crystals 2018, 8(2), 62; https://doi.org/10.3390/cryst8020062

Characterization of the PB2 Cap Binding Domain Accelerates Inhibitor Design

1
Department of Chemistry, Georgia State University, Atlanta, GA 30302, USA
2
Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30302, USA
3
State Key Lab of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
4
Department of Biochemistry and Molecular Biology, School of Life Sciences, Peking University, Beijing 100871, China
*
Author to whom correspondence should be addressed.
Received: 7 December 2017 / Revised: 16 January 2018 / Accepted: 23 January 2018 / Published: 28 January 2018
(This article belongs to the Special Issue Recent Advances in Protein Crystallography)
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Abstract

X-ray crystallographic structural determinations of the PB2 cap binding domain (PB2cap) have improved the conformational characterization of the RNA-dependent RNA polymerase machinery (PA, PB2, and PB1) of the influenza virus. Geometrically, the catalytic PB1 subunit resembles the palm of a human hand. PA lies near the thumb region, and PB2 lies near the finger region. PB2 binds the cap moiety in the pre-mRNA of the host cell, while the endonuclease of PA cleaves the pre-mRNA 10–13 nucleotides downstream. The truncated RNA piece performs as a primer for PB1 to synthesize the viral mRNA. Precisely targeting PB2cap with a small molecule inhibitor will halt viral proliferation via interference of the cap-snatching activity. Wild-type and mutant PB2cap from A/California/07/2009 H1N1 were expressed in Escherichia coli, purified by nickel affinity and size exclusion chromatography, crystallized, and subjected to X-ray diffraction experiments. The crystal of mutant PB2cap liganded with m7GTP was prepared by co-crystallization. Structures were solved by the molecular replacement method, refined, and deposited in the Protein Data Bank (PDB). Structural determination and comparative analyses of these structures revealed the functions of Glu361, Lys376, His357, Phe404, Phe323, Lys339, His432, Asn429, Gln406, and Met401 in PB2cap, and the dissociation of the influenza A PB2cap C-terminal subdomain (residues 446–479) upon ligand binding. Understanding the role of these residues will aid in the ultimate development of a small-molecule inhibitor that binds both Influenza A and B virus PB2cap. View Full-Text
Keywords: cap-snatching; polymerase basic protein 2; RNA polymerase; domain movement cap-snatching; polymerase basic protein 2; RNA polymerase; domain movement
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Constantinides, A.E.; Severin, C.C.; Gumpper, R.H.; Zheng, X.; Luo, M. Characterization of the PB2 Cap Binding Domain Accelerates Inhibitor Design. Crystals 2018, 8, 62.

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