Characterization of the PB2 Cap Binding Domain Accelerates Inhibitor Design

Xiaofeng Zheng,Ryan Gumpper,Amanda Constantinides,Chelsea Severin,Ming Luo

doi:10.3390/cryst8020062

Xiaofeng Zheng, Ryan Gumpper + Show 3 more

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https://doi.org/10.3390/cryst8020062

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Journal: Crystals	Publication Date: Jan 28, 2018
Citations: 3	License type: CC BY 4.0

Affiliation: Peking University, Georgia State University

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.

Highlights

The influenza virus continues to have a devastating impact on the global population as it evolves in defiance of current countermeasures
For the structure determination of the wild-type (PDB code: 5EG8), m7 GTP-mutant (PDB code: 5EG7), and mutant CA09-PB2cap (PDB code: 5WOP) (Table 1), the A/California/07/2009 H1N1 DNA coding sequence was first inserted between NdeI and Xho1 restrictions sites into the pET28a expression vector to generate a His6 -tag chimeric protein [10]
We suggest that when Additional the cap binds in the the induced conformational change results that these morphologies are present in other H1N1 PB2cap structures (Figures 5c,d, 6, and Table 2)

Summary

Introduction

The influenza virus continues to have a devastating impact on the global population as it evolves in defiance of current countermeasures. Adamantane derivatives, which were once effective against the influenza A virus, have lost their clinical practicality and potency against current strains of the virus. A new generation of antiviral drugs, neuraminidase inhibitors, has recently been developed as effective treatment against infection by influenza A and B viruses. The rate of developing resistance against this class of inhibitors is of critical importance as single-molecule inhibition strategies may fail. The inspiration for a novel small-molecule inhibitor originates from the difference between cap-binding patterns of human and influenza proteins. 7-methyl-G(50 )ppp(50 )N at the 50 end of human capped pre-mRNA must first be recognized by the eukaryotic initiation factor

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Conclusion