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. 2008 Oct;28(19):5965-76.
doi: 10.1128/MCB.00761-08. Epub 2008 Aug 4.

Crystal structure of human Edc3 and its functional implications

Sharon H M Ling  1 , Carolyn J Decker, Martin A Walsh, Meipei She, Roy Parker, Haiwei Song
Affiliations

Crystal structure of human Edc3 and its functional implications

Sharon H M Ling et al. Mol Cell Biol. 2008 Oct.

Abstract

Edc3 is an enhancer of decapping and serves as a scaffold that aggregates mRNA ribonucleoproteins together for P-body formation. Edc3 forms a network of interactions with the components of the mRNA decapping machinery and has a modular domain architecture consisting of an N-terminal Lsm domain, a central FDF domain, and a C-terminal YjeF-N domain. We have determined the crystal structure of the N-terminally truncated human Edc3 at a resolution of 2.2 A. The structure reveals that the YjeF-N domain of Edc3 possesses a divergent Rossmann fold topology that forms a dimer, which is supported by sedimentation velocity and sedimentation equilibrium analysis in solution. The dimerization interface of Edc3 is highly conserved in eukaryotes despite the overall low sequence homology across species. Structure-based site-directed mutagenesis revealed dimerization is required for efficient RNA binding, P-body formation, and likely for regulating the yeast Rps28B mRNA as well, suggesting that the dimeric form of Edc3 is a structural and functional unit in mRNA degradation.

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Figures

FIG. 1.
FIG. 1.
hEdc3 proteins form dimers in solution. (A) Domain architecture of the hEdc3 protein, showing the N-terminal Lsm domain, a central FDF domain, the C-terminal YjeF-N domain, and a putative low-complexity linker region between the Lsm and FDF domains. Numbers below the schematic protein outline represent the amino acid positions for the domain boundaries. (B and C) hEdc3-250C wild type (B) and hEdc3-250C E306A V310A (C) were analyzed by sedimentation equilibrium and fitted to an ideal single-species model. Representative fits for each protein are shown. (D) The graph shows the molar mass distribution, c(M), of the hEdc3-250C wild-type and hEdc3-250C E306A V310A mutant proteins. (E) The graph shows the molar mass distribution, c(M), of hEdc3FL wild-type and hEdc3FL E306A V310A mutant proteins.
FIG. 2.
FIG. 2.
Structure of hEdc3-250C and comparison of it with other YjeF-N proteins. (A) Ribbon diagram of the hEdc3-250C homodimer formed by molecules A (green) and B (yellow). Secondary structure elements are labeled. (B) Diagram of superimposed structures of hEdc3-250C (cyan) and AI-BP (PDB 2o8n; magenta). (C) Diagram of superimposed structures of hEdc3-250C (cyan) and a hypothetical protein (TM0922) from Thermotoga maritima (PDB 2ax3) (wheat colored). (D) Diagram of superimposed structures of hEdc3-250C (cyan) and a yeast hypothetical protein, YNL200C (PDB 1jzt) (gray). (E) Diagram of superimposed structures of hEdc3-250C (cyan) and methylene tetrahydromethanopterin dehydrogenase (MtdA) (PDB 1lua) (orange). The view of hEdc3-250C is the same in panels B, C, D, and E.
FIG. 3.
FIG. 3.
Surface view of hEdc3 and its structural homologs, showing the putative ligand binding pockets. (A) MtdA (orange) with bound NADP (in stick model). (B) AI-BP (magenta) with a bound sulfate ion (in stick model) in the putative binding pocket. (C) YNL200C (gray) with a bound chloride ion (in yellow dot model). (D) The YjeF-N domain of TM0922 (in wheat) with a bound glycerol molecule (in stick model). (E) hEdc3-250C (cyan) with a close-up view showing the residues located in the pocket.
FIG. 4.
FIG. 4.
Dimer interface of the YjeF-N domain of hEdc3-250C. (A) Stereo diagram with the YjeF-N domain dimer interface of hEdc3-250C is shown in green for chain A and yellow for chain B. Residues involved in dimerization are shown in stick models and labeled in dark green and brown for chains A and B. (B) Alignment of amino acid sequences of human, mouse, chicken, zebrafish, and S. cerevisiae Edc3 proteins. The secondary structures shown above the sequences are for hEdc3. Mutated residues involved in YjeF-N domain dimerization are marked with an asterisk. Invariant residues are represented in white letters on a red background, similar residues are in black with bold and unbold letters on a yellow background, and others are in black and unbold letters. Residues with similarities between 0.7 and 1.0 as defined in EScript (24) are boxed in blue. (C) Sensorgram showing single-stranded RNA binding activity of 400 nM hEdc3FL, hEdc3FL E306A V310A, hEdc3-N250, hEdc3-N236, hEdc3-203C, and hEdc3-250C.
FIG. 5.
FIG. 5.
Effect of point mutations in the dimer interface of the YjeF-N domain on the two-hybrid interaction of Edc3 with itself and on P-body aggregation in S. cerevisiae. (A) Two-hybrid interaction of Gal4 binding domain (BD) fusions of wild-type full-length Edc3p, Edc3p lacking the C-terminal Yjef-N domain (16), and the R360A H361A mutant of Edc3p with Gal4 activation domain (AD) fusions of full-length wild-type Edc3p, the catalytic domain of Dcp2p, and the activation domain alone, assayed by growth on plates containing 100 mM 3-aminotriazole. (B) Western analysis showing the levels of the Flag-tagged R360A H361A version of Edc3p relative to wild-type Flag-tagged Edc3p in edc3Δ lsm4ΔC cells. (C) Localization of GFP-tagged proteins to P-bodies after 10 min of glucose depletion in strains lacking Edc3p and the C-terminal domain of Lsm4p (edc3Δ lsm4ΔC) or Edc3p alone (edc3Δ) expressing wild-type or mutant Flag-tagged versions of Edc3p. The average number of GFP foci per cell was 3.0 ± 0.0 in the wild type and 1.1 ± 0.9 in the R360A H361A mutant for Dcp2GFP, 0.7 ± 0.5 in the wild type and 0.4 ± 0.3 in the R360A H361A mutant for Pat1GFP, 1.9 ± 0.7 in the wild type and 0.2 ± 0.3 in the R360A H361A mutant for Lsm1GFP, and 2.4 ± 0.2 in the wild type and 0.7 ± 0.2 in the R360A H361A mutant for Xrn1GFP.
FIG. 6.
FIG. 6.
Effect of deletion of Edc3p domains and point mutations in the dimer interface of the YjeF-N domain on Rps28B mRNA levels in S. cerevisiae. Northern analysis results show Rps28B mRNA levels in the edc3Δ lsm4ΔC strain with no Edc3p, wild-type Flag-tagged Edc3p, or mutant versions of Edc3p expressed from a centromere plasmid. Scr1RNA, the RNA component of the signal recognition particle, was used as a loading control.
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