Crystal structure of the human PRMT5:MEP50 complex Stephen Antonysamya,1, Zahid Bondayb, Robert M. Campbellb, Brandon Doyleb, Zhanna Druzinaa, Tarun Gheyia, Bomie Hanb, Louis N. Jungheimb, Yuewei Qianb, Charles Raucha, Marijane Russella, J. Michael Saudera, Stephen R. Wassermanc, Kenneth Weicherta, Francis S. Willardb, Aiping Zhanga, and Spencer Emtagea,1 aLilly Biotechnology Center, Eli Lilly and Company, San Diego, CA 92121; bLilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285; and cLilly Research Laboratories Collaborative Access Team, Eli Lilly and Company, Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439 Edited* by Wayne A. Hendrickson, Columbia University, New York, NY, and approved September 20, 2012 (received for review June 12, 2012) Protein arginine methyltransferases (PRMTs) play important roles RAS to ERK signaling pathway through methylation of RAF in several cellular processes, including signaling, gene regulation, proteins (10), regulates ribosome biogenesis through methylation and transport of proteins and nucleic acids, to impact growth, of ribosomal protein S10 (RPS10) (11), and plays an essential role differentiation, proliferation, and development. PRMT5 symmetri- in cell survival through regulation of eIF4E expression and cally di-methylates the two-terminal ω-guanidino nitrogens of ar- p53 translation. PRMT5 in association with MEP50 methylates ginine residues on substrate proteins. PRMT5 acts as part of a mul- cytosolic H2A to repress differentiation genes in ES cells (12). timeric complex in concert with a variety of partner proteins that During germ-cell development, the PRMT5 complex with tran- regulate its function and specificity. A core component of these scriptional repressor Blimp1 translocates from the nucleus to the complexes is the WD40 protein MEP50/WDR77/p44, which medi- cytoplasm at embryonic day 11.5 (E11.5), concomitantly with the ates interactions with binding partners and substrates. We have up-regulation of likely target genes (13). Recent data indicate that PRMT5 and MEP50 are components of the Grg4 complex, es- determined the crystal structure of human PRMT5 in complex with sential for its mediation of transcriptional repression (14). PRMT5 MEP50 (methylosome protein 50), bound to an S-adenosylmethio- has also been shown to inhibit the tumor suppressive function of nine analog and a peptide substrate derived from histone H4. The PDCD4 (15). The methyltransferase activity of PRMT5 can be structure of the surprising hetero-octameric complex reveals the controlled by phosphorylation of either MEP50 or of PRMT5 β close interaction between the seven-bladed -propeller MEP50 itself. Cyclin D1/CDK4 phosphorylates Thr5 on MEP50, activating and the N-terminal domain of PRMT5, and delineates the structural the methyltransferase activity of PRMT5 and resulting in pro- elements of substrate recognition. longed survival of tumor cells (16). In contrast, oncogenic mutants of Jak2 (V617F, K539L) phosphorylate tyrosine residues 297, 304, epigenetics | protein-protein complex | A9145C and 306 of PRMT5, disrupt association with MEP50 and down- regulate its methyltransferase activity on histone substrates (17). osttranslational methylation of lysine and arginine residues MEP50 was initially identified as a WD40 repeat protein that Pby protein lysine methyltransferases and protein arginine associates with PRMT5 and as an integral component of the 20S methyltransferases (PRMTs) alters the activity and interactions protein methyltransferase complex, termed the methylosome of substrate proteins, with crucial consequences to diverse cellular (18), and independently as an androgen receptor cofactor (p44) functions (1–3). Histone methylation is an epigenetic mark that that is overexpressed in prostate cancer cells (19). WD40 pro- plays a vital role in normal cell function, and whose dysregulation teins are known to play vital roles in various cellular networks – is associated with several diseases (4). (20, 21). These proteins function as protein protein and pro- The PRMT family of methyltransferases belongs to the largest tein–DNA interaction platforms and also as recognition mod- fi class (class I) of S-adenosylmethionine (AdoMet)-dependent meth- ules of posttranslational modi cations (22, 23). Recent studies fi yltransferase enzymes, responsible for the transfer of a methyl group on MEP50 identi ed the presence of two nuclear exclusion from AdoMet to the arginine side-chains of histones and other signals and three nuclear localization signals that control its proteins. PRMTs are further subdivided into type I, type II, type subcellular localization and its function as a transcriptional III, and type IV enzymes based on their patterns of arginine cofactor of androgen receptor during prostate development and methylation. Eleven human PRMTs have been identified to date tumorigenesis (24). MEP50 serves as a coactivator of both an- (5), and they all methylate the terminal guanidino nitrogen atoms drogen receptor and estrogen receptor in ovarian cells, and of arginine residues. Type I PRMT enzymes (PRMT1, -2, -3, -4, mediates hormonal effects during ovarian tumorigenesis (25). -6, and -8) generate ω-NG-monomethyl and ω-NG,NG-asymmetric MEP50 has been shown to interact with SUZ12 (a component di-methyl arginines, whereas PRMT5 is a type II PRMT that cat- of the PRC2/EED-EZH2 complexes), and to selectively bind alyzes the formation of ω-NG-monomethyl and ω-NG,N′G-sym- H2A, and has been postulated to be an adapter protein between PRMT5 and its substrates (26). MEP50 and PRMT5 have been metric di-methyl arginine residues. PRMT7 was initially thought to fi have type II activity, but recent evidence suggests that it may be identi ed as components of the FCP1 complex, suggesting a link a type III enzyme that is only able to monomethylate substrates to form ω-NG-monomethyl arginine (6). A type IV enzyme that ca- talyses the formation of δ-N-methyl arginine has been identified in Author contributions: S.A., Z.D., T.G., B.H., C.R., M.R., J.M.S., F.S.W., and S.E. designed yeast (7). All PRMTs share the highly conserved methyltransferase research; S.A., B.D., Z.D., T.G., B.H., C.R., M.R., J.M.S., S.R.W., K.W., F.S.W., and A.Z. performed research; L.N.J. and Y.Q. contributed new reagents/analytic tools; S.A., B.D., catalytic domain, and several PRMTs contain additional domains Z.D., T.G., B.H., C.R., M.R., S.R.W., F.S.W., A.Z., and S.E. analyzed data; and S.A., Z.B., R.M.C., that modulate their activity and specificity. PRMT2, PRMT3, and B.D., Z.D., B.H., J.M.S., F.S.W., and S.E. wrote the paper. fi PRMT9 contain SH3, zinc nger, and TRP2 domains, respectively, Conflict of interest statement: This research was funded by Lilly Research Laboratories and PRMT5 contains a largely uncharacterized N-terminal region. and all the authors are employees of Eli Lilly and Company. In contrast to type I PRMTs, PRMT5 functions as part of various *This Direct Submission article had a prearranged editor. high molecular weight protein complexes that invariably contain Freely available online through the PNAS open access option. the WD-repeat–containing protein MEP50 (methylosome protein Data deposition: The atomic coordinates and structure factors have been deposited in the 50). PRMT5 associates with other cellular proteins in a context- Research Collaboratory for Structural Bioinformatics Protein Data Bank, www.rscb.org dependent manner (Fig. S1), enabling the methylation of a myriad (PDB ID code 4GQB). of cytoplasmic and nuclear substrates, including Sm proteins, 1To whom correspondence may be addressed. E-mail: [email protected] or nucleolin, p53, histones H2A, H3, and H4, SPT5, and MBD2, and [email protected]. thereby plays a role in RNA processing, chromatin remodeling, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and control of gene expression (1, 8, 9). PRMT5 modulates the 1073/pnas.1209814109/-/DCSupplemental. 17960–17965 | PNAS | October 30, 2012 | vol. 109 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1209814109 Downloaded by guest on September 30, 2021 between transcription elongation and splicing (27, 28). PRMT5 that eluted as a dimer in gel filtration columns, but had a pro- together with MEP50 is thought to constitute a core complex, pensity to aggregate and did not crystallize. which either binds pICln to methylate Sm proteins, or binds Coexpression of PRMT5 with MEP50 in insect cells increased Riok1 to methylate nucleolin (29, 30). the yield of soluble protein, and dramatically improved the ho- Protein methyltransferases are being actively pursued as drug mogeneity of the purified product, as judged by gel filtration (Fig. targets for various types of cancer where enhanced levels of 1 A–C and Fig. S2). In contrast to the homo-dimer observed from PRMT5 have been observed (4, 31–33). Despite the vital role PRMT5 alone, coexpression with MEP50 produced a tight com- played by PRMT5 in diverse cellular processes, and its potential plex that eluted from preparative gel filtration columns in the 400- as a drug target, there has been scant structural information on to 500-kDa molecular weight range. To additionally characterize PRMT5 or its interaction with MEP50. We present here the this complex, we determined the molecular weight by sedimenta- complex of human PRMT5 with MEP50 analyzed by chroma- tion velocity analytical centrifugation (Fig. 1D). The most abun- tography, sedimentation analysis, enzymology, and X-ray crystal- dant species (65% by mass) had an estimated molecular weight of lography. We have elucidated the crystal structure of the complex 435 kDa, consistent with a PRMT5:MEP50 complex containing bound to an AdoMet analog A9145C (34) and a substrate peptide four molecules each of PRMT5 and MEP50. The second most from histone H4. As we were preparing this article, the crystal abundant species (12.5% by mass), with an estimated molecular structure of PRMT5 from Caenorhabditis elegans, which shares weight of 720 kDa, potentially consists of higher-order complexes a sequence identity of 31% with human PRMT5, was published of hetero-octamer, or alternative PRMT5:MEP50 complexes.