Journal: Nucleic Acids Res / Year: 2024 Title: The missing part: the Archaeoglobus fulgidus Argonaute forms a functional heterodimer with an N-L1-L2 domain protein. Authors: Elena Manakova / Edvardas Golovinas / Reda Pocevičiūtė / Giedrius Sasnauskas / Arunas Silanskas / Danielis Rutkauskas / Marija Jankunec / Evelina Zagorskaitė / Edvinas Jurgelaitis / ...Authors: Elena Manakova / Edvardas Golovinas / Reda Pocevičiūtė / Giedrius Sasnauskas / Arunas Silanskas / Danielis Rutkauskas / Marija Jankunec / Evelina Zagorskaitė / Edvinas Jurgelaitis / Algirdas Grybauskas / Česlovas Venclovas / Mindaugas Zaremba Abstract: Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid ...Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid targets and are responsible for gene expression regulation, mobile genome element silencing, and defence against viruses or plasmids. According to their domain organization, Agos are divided into long and short Agos. Long Agos found in prokaryotes (long-A and long-B pAgos) and eukaryotes (eAgos) comprise four major functional domains (N, PAZ, MID and PIWI) and two structural linker domains L1 and L2. The majority (∼60%) of pAgos are short pAgos, containing only the MID and inactive PIWI domains. Here we focus on the prokaryotic Argonaute AfAgo from Archaeoglobus fulgidus DSM4304. Although phylogenetically classified as a long-B pAgo, AfAgo contains only MID and catalytically inactive PIWI domains, akin to short pAgos. We show that AfAgo forms a heterodimeric complex with a protein encoded upstream in the same operon, which is a structural equivalent of the N-L1-L2 domains of long pAgos. This complex, structurally equivalent to a long PAZ-less pAgo, outperforms standalone AfAgo in guide RNA-mediated target DNA binding. Our findings provide a missing piece to one of the first and the most studied pAgos.
Mass: 18.015 Da / Num. of mol.: 265 / Source method: isolated from a natural source / Formula: H2O
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Details
Has ligand of interest
N
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Experimental details
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Experiment
Experiment
Method: X-RAY DIFFRACTION / Number of used crystals: 1
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Sample preparation
Crystal
Density Matthews: 2.47 Å3/Da / Density % sol: 50.12 %
Crystal grow
Temperature: 280 K / Method: vapor diffusion, sitting drop Details: Protein concentration 4.1 mg/ml. Reservoir: TrisHCl pH 8.0 0.05 M; iPrOH 27%; ammonium acetate 0.11 M; Bicine pH 9.0 0.05 M. Cryo-protection: short wash in 0.16 M Ammonium acetate; 0.08 M ...Details: Protein concentration 4.1 mg/ml. Reservoir: TrisHCl pH 8.0 0.05 M; iPrOH 27%; ammonium acetate 0.11 M; Bicine pH 9.0 0.05 M. Cryo-protection: short wash in 0.16 M Ammonium acetate; 0.08 M TrisHCl pH 8.5; 24% iPrOH; Glycerol 20% PH range: 8-9
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Data collection
Diffraction
Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source
Source: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 0.9797 Å
Detector
Type: DECTRIS PILATUS3 X 6M / Detector: PIXEL / Date: Nov 23, 2018
Radiation
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
Wavelength: 0.9797 Å / Relative weight: 1
Reflection
Resolution: 1.4→94.72 Å / Num. obs: 61624 / % possible obs: 100 % / Redundancy: 9.7 % / Biso Wilson estimate: 19.676 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.047 / Rpim(I) all: 0.016 / Rrim(I) all: 0.049 / Χ2: 0.97 / Net I/av σ(I): 6.6 / Net I/σ(I): 23.8
Method to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.4→53.7 Å / Cor.coef. Fo:Fc: 0.977 / Cor.coef. Fo:Fc free: 0.968 / Cross valid method: THROUGHOUT / ESU R: 0.056 / ESU R Free: 0.054 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.17772
6208
10.1 %
RANDOM
Rwork
0.14458
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obs
0.1479
55371
99.99 %
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Solvent computation
Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK