Bacterium Thermus thermophilus Argonaute (Ago; TtAgo) is a prokaryotic Ago (pAgo) that acts as the host defense against the uptake and propagation of foreign DNA by catalyzing the DNA cleavage reaction. The TtAgo active site consists of a plugged-in glutamate finger with two arginine residues (R545 and R486) located symmetrically around it. An interesting challenge is to understand how they can collaboratively facilitate enzymatic catalysis. In Kluyveromyces polysporus Ago, a eukaryotic Ago, the evolutionarily symmetrical residues are arginine and histidine, both of which function to stabilize the plugged-in catalytic tetrad conformation. Surprisingly, our simulation results indicated that, in TtAgo, only R545 is involved in the cleavage reaction by serving as a critical structural anchor to stabilize the catalytic tetrad Asp-Glu-Asp-Asp that is completed by the insertion of the glutamate finger, whereas R486 is not involved in target cleavage. The TtAgo-mediated target DNA cleavage occurs in a substrate-assisted mechanism, in which the pro-Rp (Rp, a tetrahedral phosphorus center with "R-type" chirality) oxygen of scissile phosphate acts as a general base to activate the nucleophilic water. Our unexpected theoretical findings on distinct roles played by R545 and R486 in TtAgo catalysis have been validated by single-point site-mutagenesis experiments, wherein the target cleavage is abolished for all mutants of R545. In sharp contrast, the cleavage activity is maintained for all mutants of R486. Our work provides mechanistic insights on the catalytic specificity of Ago proteins and could facilitate the design of new gene-editing tools in the long term.
Bibliographical noteKAUST Repository Item: Exported on 2020-10-01
Acknowledged KAUST grant number(s): OSR-2016-CRG5-3007
Acknowledgements: This work was supported by Hong Kong Research Grant Council (Hong Kong University of Science and Technology) Grants C6009-15G, 16318816, 16302214, AoE/P-705/16, and T31-605/18-W; King Abdullah University of Science and Technology (KAUST) Office of Sponsored Research (OSR) Grant OSR-2016-CRG5-3007; Innovation and Technology Commission Grants ITCPD/17-9 and ITC-CNERC14SC01; National Natural Science Foundation of China Grants 31725008, 31571335, and 31630015; and National Institutes of Health Grant R35-GM127040. This research made use of the resources of the Supercomputing Laboratory at KAUST. X.H. is the Padma Harilela Associate Professor of Science.