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Structural insights into protein arginine symmetric dimethylation by PRMT5 - PubMed

  • ️Sat Jan 01 2011

Structural insights into protein arginine symmetric dimethylation by PRMT5

Litao Sun et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Symmetric and asymmetric dimethylation of arginine are isomeric protein posttranslational modifications with distinct biological effects, evidenced by the methylation of arginine 3 of histone H4 (H4R3): symmetric dimethylation of H4R3 leads to repression of gene expression, while asymmetric dimethylation of H4R3 is associated with gene activation. The enzymes catalyzing these modifications share identifiable sequence similarities, but the relationship between their catalytic mechanisms is unknown. Here we analyzed the structure of a prototypic symmetric arginine dimethylase, PRMT5, and discovered that a conserved phenylalanine in the active site is critical for specifying symmetric addition of methyl groups. Changing it to a methionine significantly elevates the overall methylase activity, but also converts PRMT5 to an enzyme that catalyzes both symmetric and asymmetric dimethylation of arginine. Our results demonstrate a common catalytic mechanism intrinsic to both symmetric and asymmetric arginine dimethylases, and show that steric constrains in the active sites play an essential role in determining the product specificity of arginine methylases. This discovery also implies a potentially regulatable outcome of arginine dimethylation that may provide versatile control of eukaryotic gene expression.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Structural and functional conservation of PRMT5. (A) A schematic representation of domain structures of C. elegans and human PRMT5, and a representative type-I arginine methylase, PRMT1 of rat. The lengths of the boxes are approximately drawn in scale with the protein lengths, and the residue numbers at domain boundaries are labeled. Areas filled in tan, cyan, green, and yellow represent TIM-barrel, Rossmann-fold, β-barrel, and oligomerization domains, respectively. Levels of amino acid identity and similarity of the individual domains between C. elegans and human PRMT5s, and that between human PRMT5 and rat PRMT1 are shown. (B) Sequence alignment. The full-length sequences of C. elegans and human PRMT5s, and the regions of the solved structures of rat PRMT1 and mouse CARM1 are aligned. Residues conserved in all four proteins are shown in white letters over purple background, and similar residues are indicated with red letters. Residues conserved in PRMT5 proteins and type-I arginine methylases are highlighted tan and yellow, respectively. Blue stars mark the CePRMT5 residues subjected to mutagenesis. At the top of the sequences, a schematic representation of the secondary structure elements of CePRMT5 is shown. Every ten residues are indicated with a “·” sign. (C) Enzymatic activity assay. Top box, coomassie-stained gel of enzymes and substrate (histone H4) used. GST-tagged rat PRMT1 and poly(His)-tagged C. elegans PRMT5 were expressed in E. coli, and flag-tagged human PRMT5 was purified from HEK293 cells. Approximately 5 μg of enzymes and histone H4 each were used in the assay. Top 2nd box, autoradiograph generated with the use of 0.25 mCi of SAM with tritiated methyl group. Top 3rd box, Western blot detection of asymmetrically dimethylated histone H4R3. Bottom box, Western blot detection of symmetrically dimethylated histone H4R3.

Fig. 2.
Fig. 2.

Overall structure of PRMT5. (A) A ribbon representation of PRMT5 monomer. Domains are colored as in Fig. 1A. Helices and strands are labeled TA to TH and T1 to T8, respectively, for the TIM-barrel domain; αA to αF and β1 to β5, respectively, for the Rossmann-fold domain; and OA-OB and O1-O2, respectively, for the oligomerization domain. Strands in the β-barrel domain are labeled b1 to b10. L0 indicates the N-terminal loop of the Rossmann-fold domain, and the SAH molecule is shown in a stick model. (B) A PRMT5 dimer is shown as a ribbon model superimposed onto a surface representation. The surface for one monomer is colored light green and the other in light blue. The red line approximately traces the dimeric interface.

Fig. 3.
Fig. 3.

Structural features of the PRMT5 active site. (A) Structural comparison with type-I arginine methylases PRMT1 (Pdb id: 1OR8; magenta) and CARM1 (Pdb id: 2V74; light blue). For visual clarity, only three regions of major differences, enclosed in red circles and labeled I, II and III, are superimposed onto the structure of PRMT5. (B) An up-close view of the active site. Key residues of PRMT5 (carbon: yellow; oxygen: red; nitrogen: blue) and the SAH molecule (carbon: orange; sulfur: gold) are shown in a stick model superimposed with a ribbon representation of PRMT5. The double-E loop of PRMT1 (while ribbon), Glu153 on it, and the bound substrate arginine (stick model; carbon: white) are also shown. (C) Methylase activities of Phe379 mutants. Top box: coomassie-stained gel of enzymes and substrate used. Bottom box: autoradiography detection. (D) Circular dichroism spectra of the wild-type and Phe379 mutants of PRMT5.

Fig. 4.
Fig. 4.

Enzymatic properties of the F379M mutant. (A) Top box: coomassie-stained gel showing varying amounts of the wide-type and the F379M mutant of PRMT5, and a constant amount of histone H4 (5.0 μg) used for enzymatic assay. “C” indicates no enzyme added. Bottom box: autoradiography detection with 0.25 mCi of [3H]-SAM used in each reaction. (B) Western blot detection of asymmetric (middle box) and symmetric (bottom box) dimethylation of histone H4R3. Top box is a coomassie-blue stained gel showing proteins used in the activity assays. Please note, for a comparable level of Werstern blot signal, the amount of the F379M protein is adjusted to ∼1/10 of the wild-type protein, and the amount of PRMT1 is even smaller. (C) Double reciprocal plot analysis of the wild-type and F379 mutant of PRMT5. Derived kinetic parameters are tabulated. (D) Conserved property of the Phe-to-Met mutants of human and nematode PRMT5s. Top two boxes: coomassie staining of the wild-type and mutant enzymes and the substrate used. The right pointing arrow indicates the position of human PRMT5 proteins; an asterisk indicates the position of rat PRMT1; and the left pointing arrow marks the position of C. elegans PRMT5. Third box: Western blot detection of the flag-tagged wild-type and mutant human PRMT5. Fourth box: Western blot detection of asymmetrically dimethylated H4R3. Bottom box: Western blot detection of symmetrically dimethylated H4R3.

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References

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