Structural insights into protein arginine symmetric dimethylation by PRMT5 Litao Suna,b,1,MingzhuWanga,1, Zongyang Lva,b, Na Yanga,YingfangLiua, Shilai Baoc,WeiminGonga,2, and Rui-Ming Xua,2 aNational Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; bGraduate University of Chinese Academy of Sciences, Beijing 100049, China; and cState Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China Edited by* Dinshaw J. Patel, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved October 19, 2011 (received for review May 2, 2011) Symmetric and asymmetric dimethylation of arginine are isomeric enzyme shares high sequence homology with its human counter- protein posttranslational modifications with distinct biological part, and the recombinant protein displays robust and specific effects, evidenced by the methylation of arginine 3 of histone H4 symmetric arginine dimethylase activity in vitro (Fig. 1). The (H4R3): symmetric dimethylation of H4R3 leads to repression of structure shows the PRMT5 is composed of four clearly defined gene expression, while asymmetric dimethylation of H4R3 is asso- domains, a previously unsuspected TIM-barrel at the N-terminal ciated with gene activation. The enzymes catalyzing these modifi- end, a middle Rossmann-fold domain, a C-terminal β-barrel cations share identifiable sequence similarities, but the relationship domain, and a ∼60 residue dimerization domain inserted be- between their catalytic mechanisms is unknown. Here we analyzed tween β1 and β2 of the β-barrel domain (Fig. 2A). The first three the structure of a prototypic symmetric arginine dimethylase, domains are packed in a triangular manner, with direct contacts PRMT5, and discovered that a conserved phenylalanine in the between sequential domains, and the oligomerization domain active site is critical for specifying symmetric addition of methyl bridges the TIM-barrel and β-barrel domains. The structure of groups. Changing it to a methionine significantly elevates the the SAH-bound PRMT5 differs from that of free protein in overall methylase activity, but also converts PRMT5 to an enzyme that a N-terminal loop (L0) and helix (αA) are ordered in the that catalyzes both symmetric and asymmetric dimethylation of SAH-bound structure. We will use the SAH-bound structure for arginine. Our results demonstrate a common catalytic mechanism analysis unless explicitly noted. BIOCHEMISTRY intrinsic to both symmetric and asymmetric arginine dimethylases, PRMT5 exists as a homodimer, shown both in the crystal struc- and show that steric constrains in the active sites play an essential ture and in solution, as determined by analytic ultracentrifugation role in determining the product specificity of arginine methylases. (Fig. 2B, Fig. S1). The dimeric interface buries a total pair wise 2 This discovery also implies a potentially regulatable outcome of surface area of 2;305 Å , and the intermolecular interactions arginine dimethylation that may provide versatile control of eukar- occur between the dimerization domains and that between the yotic gene expression. TIM-barrel and β-barrel domains. Human PRMT5 has a shorter oligomerization domain, but extensive conservation of amino histone methylation ∣ transtriptional regulation ∣ RNA splicing ∣ acids involved in intermolecular interactions implies that it also crystal structure forms a dimer, consistent with the report of dimeric and higher oligomeric forms of human PRMT5 (5). In fact, all arginine methy- rotein arginine methyltransferase 5 (PRMT5) catalyzes the lases with known structures dimerize via a homologous dimeriza- Pevenly addition of two methyl groups to the two ω-guanidino tion domain, also known as the dimerization “arm” (Fig. 3A), G 0G nitrogen atoms of arginine, resulting in ω-N , N symmetric (22–26). Thus, protein dimerization appears to be an evolutionarily dimethylation of arginine (sDMA) of the target protein (1–5). conserved property of arginine methylases, although the functional PRMT5 functions in the nucleus as well as in the cytoplasm, significance remains poorly understood. and its substrates include histones, spliceosomal proteins, tran- scription factors, and proteins involved in piRNA biogenesis Comparison with Arginine Asymmetric Dimethylases. The overall (6). Symmetric dimethylation of these proteins profoundly impact fold and spatial positioning of the Rossmann-fold and β-barrel many biological processes; e.g., epigenetic control of gene expres- domains are similar to that of type-I enzymes, represented by sion (7), splicing regulation (2, 3, 8, 9), circadian rhythms (9, 10), PRMT1 and CARM1 (Fig. 3A) (24–26). The root-mean-squared DNA damage response (11, 12), and germ cell development and deviation of Cα positions between PRMT5 and PRMT1 is pluripotency (13–16). Interestingly, both PRMT5 and a group of approximately 2.1 Å when both the Rossmann-fold and β-barrel asymmetric (type-I) arginine dimethylases, which add two methyl domains are compared, whereas it is 1.4 Å for the Rossmann-fold groups to the same ω-guanidino nitrogen atom (aDMA), share domain alone. In particular, a segment including a N-terminal common recognition sequences, and the target arginine can often loop (L0) and a following helix (αA) (a.a. 359–380) became be symmetrically or asymmetrically dimethylated. Yet, these iso- ordered upon SAH binding. Helix αA is positioned similar to that meric modifications have distinct biological effects. One such ex- ample occurs at arginine-3 of histone H4 (H4R3). Symmetric dimethylation of H4R3 has been linked to repression of gene Author contributions: L.S., Y.L., S.B., W.G., and R.-M.X. designed research; L.S., M.W., Z.L., – N.Y., and R.-M.X. performed research; S.B. contributed new reagents/analytic tools; L.S., expression (17 19), while asymmetric dimethylation of H4R3 is M.W., Z.L., N.Y., Y.L., S.B., W.G., and R.-M.X. analyzed data; and L.S., M.W., and R.-M.X. associated with gene activation (20, 21). The startling difference wrote the paper. in biological effects of sDMA and aDMA modifications necessi- The authors declare no conflict of interest. tates the understanding of the enzymatic mechanisms differentiat- *This Direct Submission article had a prearranged editor. ing the two chemically isomeric but functionally antagonistic Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, posttranslational modifications. www.pdb.org (PDB ID codes 3UA3 and 3UA4). 1L.S. and M.W. contributed equally to this work. Results 2To whom correspondence may be addressed. E-mail: [email protected] or wgong@ Overall Structure. We have determined the crystal structures of sun5.ibp.ac.cn. full-length PRMT5 from Caenorhabditis elegans, alone and in This article contains supporting information online at www.pnas.org/lookup/suppl/ complex with S-Adenosyl-L-homocysteine (SAH). The nematode doi:10.1073/pnas.1106946108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1106946108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 29, 2021 Fig. 1. Structural and functional conservation of PRMT5. (A) A schematic representation of domain structures of C. elegans and human PRMT5, and a re- presentative 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 symme- trically dimethylated histone H4R3. found in type-I enzymes, sheltering SAH from exposing to the PRMT5 proteins (Pro366, Leu367, and Leu371) and forms a solvent and creating a secluded catalytic active site. However, solvent inaccessible area for catalysis; (ii) the N-terminal end key residues responsible for the disordered-to-ordered conforma- of L0 makes a U-turn and contacts the dimerization domain, tional transition upon SAH/SAM binding are separately con- which stabilizes the loop in a conformation endowed with the served among PRMT5 family members (Fig. 1B). Among which, ability to influence substrate binding (Figs. 3 A and B). Hence, Tyr376 and Phe379 on αA interact with the ribose and homocys- the highly conserved PRMT5 loop is important for SAH/SAM teine moieties via hydrogen bonds and van der Waals contacts, binding, as well as in a position to regulate substrate binding. respectively. Loop L0 contains several amino acids uniquely conserved in PRMT5s across species. The corresponding region The Active Site. The active site of PRMT5 is identified by the loca- in PRMT1 is disordered, and that of CARM1 adopts a helical tion of the sulfur atom of SAH and a pair of invariant glutamate conformation.