title: Structural insights into the N-terminal GIY-YIG endonuclease activity of Arabidopsis glutaredoxin AtGRXS16 in chloroplasts.
abstract: Glutaredoxins (Grxs) have been identified across taxa as important mediators in various physiological functions. A chloroplastic monothiol glutaredoxin, AtGRXS16 from Arabidopsis thaliana, comprises two distinct functional domains, an N-terminal domain (NTD) with GlyIleTyr-TyrIleGly (GIY-YIG) endonuclease motif and a C-terminal Grx module, to coordinate redox regulation and DNA cleavage in chloroplasts. Structural determination of AtGRXS16-NTD showed that it possesses a GIY-YIG endonuclease fold, but the critical residues for the nuclease activity are different from typical GIY-YIG endonucleases. AtGRXS16-NTD was able to cleave λDNA and chloroplast genomic DNA, and the nuclease activity was significantly reduced in AtGRXS16. Functional analysis indicated that AtGRXS16-NTD could inhibit the ability of AtGRXS16 to suppress the sensitivity of yeast grx5 cells to oxidative stress; however, the C-terminal Grx domain itself and AtGRXS16 with a Cys123Ser mutation were active in these cells and able to functionally complement a Grx5 deficiency in yeast. Furthermore, the two functional domains were shown to be negatively regulated through the formation of an intramolecular disulfide bond. These findings unravel a manner of regulation for Grxs and provide insights into the mechanistic link between redox regulation and DNA metabolism in chloroplasts.
authors.affiliation: Ministry of Education Key Laboratory of Protein Science, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Liu
authors.forename: Shian
authors.initials: S
authors.lastname: Liu
authors.forename: Yingang
authors.initials: Y
authors.lastname: Feng
authors.forename: Jian-Zhong
authors.initials: JZ
authors.lastname: Liu
authors.forename: Yuling
authors.initials: Y
authors.lastname: Chen
authors.forename: Khanh
authors.initials: K
authors.lastname: Pham
authors.forename: Haiteng
authors.initials: H
authors.lastname: Deng
authors.forename: Kendal D
authors.initials: KD
authors.lastname: Hirschi
authors.forename: Xinquan
authors.initials: X
authors.lastname: Wang
authors.forename: Ninghui
authors.initials: N
authors.lastname: Cheng
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D017920:Antiporters; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology*; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D054477:Glutaredoxins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D020125:Mutation, Missense / Q000235:genetics; D010084:Oxidation-Reduction; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017382:Reactive Oxygen Species / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D015003:Yeasts
chemical_list: D017920:Antiporters; D029681:Arabidopsis Proteins; C473126:CXIP1 protein, Arabidopsis; D002352:Carrier Proteins; D054477:Glutaredoxins; C528773:Grx5 protein, S cerevisiae; D017382:Reactive Oxygen Species; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004720:Endonucleases; C582684:GRXS16 protein, Arabidopsis
keywords: nuclear magnetic resonance; reactive oxygen species