Two symmetric arginine residues play distinct roles in Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

Abstract

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.

1′-Ribose cyano substitution allows Remdesivir to effectively inhibit nucleotide addition and proofreading during SARS-CoV-2 viral RNA replication

“…To assess the stability of cleavage site, we examined the distance between MgA and the O3' atom of the second last nucleotide at the 3'-terminal as it is essential for cleavage (67-69) (Figure 5A). This distance is well maintained in majority of the simulations for the wildtype-RNA (Supplementary Figure S11), and the histogram shows the highest peak in the region of 2.0~2.5 Å (Figure 5A) suitable for cleavage (67)(68)(69). By sharp contrast, MD simulations with Remdesivir at 3'-terminal show destabilization of cleavage site in ExoN, with the increased height of the second peak at ~3.7 Å (Figure 5A and Supplementary Figure S12).…”

Expression and Functional Analysis of the Argonaute Protein of Thermus thermophilus (TtAgo) in E. coli BL21(DE3)

“…The PIWI-like domain is thought to possess the catalytic ability to degrade target DNA or RNA due to the conserved tetrad catalytic center [ 20 ]. All of the currently described dead TtAgo mutations are located in the PIWI-like domain, such as the two symmetrical arginine residues (R545, involved in the cleavage reaction, and R486, not involved in target cleavage), which play distinct roles in ssDNA target cleavage [ 48 ], and TtAgoDM (Double mutant; D478A, D546A), which showed no cleavage activity in in vitro cleavage assay [ 31 ]. Surprisingly, no mutations within the PIWI domain were isolated in our high-throughput screen.…”

“…Surprisingly, no mutations within the PIWI domain were isolated in our high-throughput screen. As controls for our experiments, we reconstructed three of these previously studied mutants in the literature [ 31 , 48 ]. These included TtAgoDM (D478A, D546A) and single mutants in the two symmetrical arginine mutations (R486H and R545H).…”

Water as a reactant in the first step of triosephosphate isomerase catalysis

“…[27][28][29][30][31] Indeed, one of the earliest instances of such a proposal grew A B from skepticism regarding Asn61 as the general base in the mechanism of RAS p21 proteins 27,[29][30][31] , a motivation that appears parallel to that pursued here. More recently are reports involving water as the ultimate catalytic base stemming from water deprotonation involving phosphate 32,33 , including computational studies that compare different detailed mechanisms, all involving proton transfer from water to phosphate at some point. 34,35 This concept does not seem to appear in previous work on TIM, however.…”