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

Jinping Lei, Gang Sheng, Peter Pak Hang Cheung, Shenglong Wang, Yu Li, Xin Gao, Yingkai Zhang, Yanli Wang, Xuhui Huang

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)845-853
Number of pages9
JournalProceedings of the National Academy of Sciences of the United States of America
Volume116
Issue number3
DOIs
StatePublished - Jan 15 2019

Fingerprint

R 486
Thermus thermophilus
DNA Cleavage
Arginine
DNA
Catalysis
Fingers
Glutamic Acid
Kluyveromyces
Histidine
Mutagenesis
Phosphorus
Catalytic Domain
Phosphates
Oxygen
Bacteria
Water

Keywords

  • Bacterial Argonaute
  • DNA cleavage
  • QM/MM simulations

ASJC Scopus subject areas

  • General

Cite this

Two symmetric arginine residues play distinct roles in thermus thermophilus argonaute DNA guide strand-mediated DNA target cleavage. / Lei, Jinping; Sheng, Gang; Cheung, Peter Pak Hang; Wang, Shenglong; Li, Yu; Gao, Xin; Zhang, Yingkai; Wang, Yanli; Huang, Xuhui.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 116, No. 3, 15.01.2019, p. 845-853.

Research output: Contribution to journalArticle

Lei, Jinping ; Sheng, Gang ; Cheung, Peter Pak Hang ; Wang, Shenglong ; Li, Yu ; Gao, Xin ; Zhang, Yingkai ; Wang, Yanli ; Huang, Xuhui. / Two symmetric arginine residues play distinct roles in thermus thermophilus argonaute DNA guide strand-mediated DNA target cleavage. In: Proceedings of the National Academy of Sciences of the United States of America. 2019 ; Vol. 116, No. 3. pp. 845-853.
@article{d466dbac53044ef6a15c87c80cabfdfa,
title = "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.",
keywords = "Bacterial Argonaute, DNA cleavage, QM/MM simulations",
author = "Jinping Lei and Gang Sheng and Cheung, {Peter Pak Hang} and Shenglong Wang and Yu Li and Xin Gao and Yingkai Zhang and Yanli Wang and Xuhui Huang",
year = "2019",
month = "1",
day = "15",
doi = "10.1073/pnas.1817041116",
language = "English (US)",
volume = "116",
pages = "845--853",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "3",

}

TY - JOUR

T1 - Two symmetric arginine residues play distinct roles in thermus thermophilus argonaute DNA guide strand-mediated DNA target cleavage

AU - Lei, Jinping

AU - Sheng, Gang

AU - Cheung, Peter Pak Hang

AU - Wang, Shenglong

AU - Li, Yu

AU - Gao, Xin

AU - Zhang, Yingkai

AU - Wang, Yanli

AU - Huang, Xuhui

PY - 2019/1/15

Y1 - 2019/1/15

N2 - 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.

AB - 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.

KW - Bacterial Argonaute

KW - DNA cleavage

KW - QM/MM simulations

UR - http://www.scopus.com/inward/record.url?scp=85060017022&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85060017022&partnerID=8YFLogxK

U2 - 10.1073/pnas.1817041116

DO - 10.1073/pnas.1817041116

M3 - Article

C2 - 30591565

AN - SCOPUS:85060017022

VL - 116

SP - 845

EP - 853

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 3

ER -