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Proc Natl Acad Sci U S A. Oct 30, 2012; 109(44): 17960–17965.
Published online Oct 15, 2012. doi:  10.1073/pnas.1209814109
PMCID: PMC3497828

Crystal structure of the human PRMT5:MEP50 complex


Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed β-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.

Keywords: epigenetics, protein-protein complex, A9145C

Posttranslational methylation of lysine and arginine residues by protein lysine methyltransferases and protein arginine methyltransferases (PRMTs) alters the activity and interactions of substrate proteins, with crucial consequences to diverse cellular functions (13). Histone methylation is an epigenetic mark that plays a vital role in normal cell function, and whose dysregulation is associated with several diseases (4).

The PRMT family of methyltransferases belongs to the largest class (class I) of S-adenosylmethionine (AdoMet)-dependent methyltransferase enzymes, responsible for the transfer of a methyl group from AdoMet to the arginine side-chains of histones and other proteins. PRMTs are further subdivided into type I, type II, type III, and type IV enzymes based on their patterns of arginine methylation. Eleven human PRMTs have been identified to date (5), and they all methylate the terminal guanidino nitrogen atoms of arginine residues. Type I PRMT enzymes (PRMT1, -2, -3, -4, -6, and -8) generate ω-NG-monomethyl and ω-NG,NG-asymmetric di-methyl arginines, whereas PRMT5 is a type II PRMT that catalyzes the formation of ω-NG-monomethyl and ω-NG,N′G-symmetric di-methyl arginine residues. PRMT7 was initially thought to have type II activity, but recent evidence suggests that it may be a type III enzyme that is only able to monomethylate substrates to form ω-NG-monomethyl arginine (6). A type IV enzyme that catalyses the formation of δ-N-methyl arginine has been identified in yeast (7). All PRMTs share the highly conserved methyltransferase catalytic domain, and several PRMTs contain additional domains that modulate their activity and specificity. PRMT2, PRMT3, and PRMT9 contain SH3, zinc finger, and TRP2 domains, respectively, and PRMT5 contains a largely uncharacterized N-terminal region.

In contrast to type I PRMTs, PRMT5 functions as part of various high molecular weight protein complexes that invariably contain the WD-repeat–containing protein MEP50 (methylosome protein 50). PRMT5 associates with other cellular proteins in a context-dependent manner (Fig. S1), enabling the methylation of a myriad of cytoplasmic and nuclear substrates, including Sm proteins, nucleolin, p53, histones H2A, H3, and H4, SPT5, and MBD2, and thereby plays a role in RNA processing, chromatin remodeling, and control of gene expression (1, 8, 9). PRMT5 modulates the RAS to ERK signaling pathway through methylation of RAF proteins (10), regulates ribosome biogenesis through methylation of ribosomal protein S10 (RPS10) (11), and plays an essential role in cell survival through regulation of eIF4E expression and p53 translation. PRMT5 in association with MEP50 methylates cytosolic H2A to repress differentiation genes in ES cells (12). During germ-cell development, the PRMT5 complex with transcriptional repressor Blimp1 translocates from the nucleus to the cytoplasm at embryonic day 11.5 (E11.5), concomitantly with the up-regulation of likely target genes (13). Recent data indicate that PRMT5 and MEP50 are components of the Grg4 complex, essential for its mediation of transcriptional repression (14). PRMT5 has also been shown to inhibit the tumor suppressive function of PDCD4 (15). The methyltransferase activity of PRMT5 can be controlled by phosphorylation of either MEP50 or of PRMT5 itself. Cyclin D1/CDK4 phosphorylates Thr5 on MEP50, activating the methyltransferase activity of PRMT5 and resulting in prolonged survival of tumor cells (16). In contrast, oncogenic mutants of Jak2 (V617F, K539L) phosphorylate tyrosine residues 297, 304, and 306 of PRMT5, disrupt association with MEP50 and down-regulate its methyltransferase activity on histone substrates (17).

MEP50 was initially identified as a WD40 repeat protein that associates with PRMT5 and as an integral component of the 20S protein methyltransferase complex, termed the methylosome (18), and independently as an androgen receptor cofactor (p44) that is overexpressed in prostate cancer cells (19). WD40 proteins are known to play vital roles in various cellular networks (20, 21). These proteins function as protein–protein and protein–DNA interaction platforms and also as recognition modules of posttranslational modifications (22, 23). Recent studies on MEP50 identified the presence of two nuclear exclusion signals and three nuclear localization signals that control its subcellular localization and its function as a transcriptional cofactor of androgen receptor during prostate development and tumorigenesis (24). MEP50 serves as a coactivator of both androgen receptor and estrogen receptor in ovarian cells, and mediates hormonal effects during ovarian tumorigenesis (25). MEP50 has been shown to interact with SUZ12 (a component of the PRC2/EED-EZH2 complexes), and to selectively bind H2A, and has been postulated to be an adapter protein between PRMT5 and its substrates (26). MEP50 and PRMT5 have been identified as components of the FCP1 complex, suggesting a link between transcription elongation and splicing (27, 28). PRMT5 together with MEP50 is thought to constitute a core complex, which either binds pICln to methylate Sm proteins, or binds Riok1 to methylate nucleolin (29, 30).

Protein methyltransferases are being actively pursued as drug targets for various types of cancer where enhanced levels of PRMT5 have been observed (4, 3133). Despite the vital role played by PRMT5 in diverse cellular processes, and its potential as a drug target, there has been scant structural information on PRMT5 or its interaction with MEP50. We present here the complex of human PRMT5 with MEP50 analyzed by chromatography, sedimentation analysis, enzymology, and X-ray crystallography. We have elucidated the crystal structure of the complex bound to an AdoMet analog A9145C (34) and a substrate peptide from histone H4. As we were preparing this article, the crystal structure of PRMT5 from Caenorhabditis elegans, which shares a sequence identity of 31% with human PRMT5, was published (35). In contrast to the dimeric C. elegans PRMT5 structure, our work reveals that human PRMT5 binds MEP50 to form a hetero-octameric complex of molecular weight ~450 kDa. This (PRMT5)4(MEP50)4 module is likely to be the core structural unit that interacts with partner proteins to form the plethora of multisubunit complexes with discrete specificities and functions.


Production and Characterization of the PRMT5:MEP50 Complex.

Initial attempts to isolate and crystallize the C-terminal catalytic domain of human PRMT5 were unsuccessful. Although it was possible to express and purify soluble constructs from Escherichia coli and insect cells, the protein was biochemically inactive and did not bind AdoMet or AdoHcy as determined by surface plasmon resonance analysis. Expression of full-length PRMT5 in insect cells yielded a soluble and biochemically active protein that eluted as a dimer in gel filtration columns, but had a propensity to aggregate and did not crystallize.

Coexpression of PRMT5 with MEP50 in insect cells increased the yield of soluble protein, and dramatically improved the homogeneity of the purified product, as judged by gel filtration (Fig. 1 A–C and Fig. S2). In contrast to the homo-dimer observed from PRMT5 alone, coexpression with MEP50 produced a tight complex that eluted from preparative gel filtration columns in the 400- to 500-kDa molecular weight range. To additionally characterize this complex, we determined the molecular weight by sedimentation velocity analytical centrifugation (Fig. 1D). The most abundant species (65% by mass) had an estimated molecular weight of 435 kDa, consistent with a PRMT5:MEP50 complex containing four molecules each of PRMT5 and MEP50. The second most abundant species (12.5% by mass), with an estimated molecular weight of 720 kDa, potentially consists of higher-order complexes of hetero-octamer, or alternative PRMT5:MEP50 complexes. There was negligible material smaller than 435 kDa, demonstrating the absence of free PRMT5 or MEP50 under these conditions. Samples were also routinely analyzed by LC-MS to identify posttranslational modifications and a fraction of MEP50 was found to be phosphorylated on Thr5 (16) (Fig. S3).

Fig. 1.
PRMT5 forms a tight complex with MEP50 when coexpressed in insect cells, and the two proteins copurify on an anti-FLAG affinity column followed by size-exclusion chromatography. (A) SDS-PAGE gel of the purified PRMT5:MEP50 complex. (B) Analytical gel ...

PRMT5:MEP50 Complex Is More Active than Dimeric PRMT5.

Samples of PRMT5 and PRMT5:MEP50 were analyzed for methyltransferase activity using a peptide derived from histone H4 (residues 1–21) as a substrate. Consistent with its description as a type II arginine methyltransferase, both PRMT5 and PRMT5:MEP50 were able to generate di-methylated H4 peptide product (Fig. 1 E and F). However, production of the di-methylated species depended critically on generation of the monomethylated peptide. The concentration of monomethylated peptide produced by PRMT5:MEP50 reached ~70 nM between 3 and 5 h (Fig. 1F), significantly exceeding the enzyme concentration of 10 nM, and production of di-methylated peptide could not be observed until the concentration of the monomethylated peptide exceeded that of the unmethylated substrate. This result demonstrates that PRMT5 has a nonprocessive enzymatic mechanism for peptide substrates, in contrast to the partially processive mechanism described for PRMT1 (36). Lack of processivity for the PRMT5 or PRMT5:MEP50 complex was observed at all tested concentrations of the peptide (0.01–10 μM) and AdoMet (0.1–10 μM).

Although there was some variability in the specific activity between different batches of enzyme preparations, the PRMT5:MEP50 complex consistently had a higher level of methyltransferase activity compared with PRMT5 alone under similar experimental conditions (Figs. 1 E and F). This apparent difference in enzyme activity was attributed to a large difference in affinities for the peptide and AdoMet between the two forms of the enzyme. The Km values of PRMT5 for the peptide and AdoMet were approximately 50- and 10-times higher, respectively, than those of the PRMT5:MEP50 complex, indicating a positive allosteric effect of MEP50 on the cofactor and substrate binding affinity of PRMT5 (Table 1). Binding of the cofactor or the substrate peptide does not appear to affect binding of the other because Km values of the AdoMet or the peptide were largely unaffected by variation in the concentration of the other (Fig. S4). The kcat value of the PRMT5:MEP50 complex was 20–25 h−1 at room temperature and a higher value was estimated for PRMT5, although accurate determination of this value for PRMT5 was difficult because of low enzyme activity and high Km values. Screening of AdoMet analogs using a scintillation proximity assay identified A9145C (34) as a potent inhibitor of PRMT5 with an IC50 of 35 nM (Fig. S5).

Table 1.
Kinetic parameters (Km and kcat values) for PRMT5 and PRMT5:MEP50 for fixed concentrations of AdoMet or substrate peptide

Overall Structure of the PRMT5:MEP50 Complex.

PRMT5:MEP50 was cocrystallized in complex with the AdoMet analog A9145C and an N-terminally acetylated substrate peptide from histone H4 (residues 1–21); X-ray diffraction data were collected at the Advanced Photon Source, Argonne, IL. Attempts at Molecular Replacement using homology models prepared from type I methyltransferases and WD40 domains were unsuccessful, leading us to determine the structure by selenomethionine (Se-Met) MAD phasing (Fig. S3). The structure revealed that the PRMT5 complex with MEP50 forms a ~453 kDa hetero-octamer in agreement with the sedimentation and chromatography data (Fig. 2A). The crystallographic asymmetric unit contains one molecule each of PRMT5 and MEP50, with the hetero-octamer being generated by the crystallographic D2 symmetry. Almost the entire PRMT5 polypeptide (residues 13–637) was ordered in the crystal structure, and residues 21–329 of MEP50 were ordered, with the exception of loops 208–211 and 245–246.

Fig. 2.
(A) Structure of the human PRMT5:MEP50 hetero-octameric complex. PRMT5 monomers-1, -2, -3, and -4 are colored green, blue, wheat, and yellow, respectively. MEP50 molecules in red decorate the outer surface of the molecule, interacting solely with the ...

PRMT5 adopts a two-domain structure, with the N-terminal domain (residues 13–292) adopting a TIM barrel structure, which makes extensive charged and hydrophobic interactions with the C-terminal catalytic domains of adjacent monomers. The PRMT5 molecules form a tetramer at the center of the complex, with MEP50 molecules decorating the outer surface and interacting solely with the TIM barrel domains of PRMT5 to form the PRMT54:MEP504 hetero-octamer. The dimerization domain in human PRMT5 is much smaller than in C. elegans (Fig. S6), and it adopts a very different fold. Residues 488–494 in this region form a short loop, with Arg488 and Asp491 from one monomer making salt bridges with Asp491 and Arg488 of an adjacent monomer (Fig. 2B). These loops from one pair of monomers pack against the equivalent loops of an adjacent pair of monomers, which are similarly linked by Arg-Asp salt bridges. Apart from this direct interaction between the catalytic domains of the four PRMT5 molecules in the hetero-octamer, the bulk of the interactions between the monomers are mediated by extensive contacts between the N-terminal TIM barrels and C-terminal catalytic domains of adjacent monomers (Fig. 2C and Fig. S7). The TIM barrel fold adopted by the N-terminal region was not predicted based on analyses of the primary sequence.

The interaction surface between the TIM barrel domain and the catalytic domain within the same monomer (buried surface area of 821 Å2) is comparable to the interactions between the monomers of the tetramer (buried surface areas of 713 and 608 Å2). The quaternary structure of one of the two dimers that make up the human PRMT5 tetramer is conserved with the C. elegans structure (Fig. S7), although the putative dimerization domains exhibit significant variability in length, conformation, and interactions between the human and C. elegans structures. Analysis of this dimer interface in the human PRMT5:MEP50 structure reveals that several of the hydrogen bonds (Asp70:OD1…Thr400:OG1, Asp70:OD2…Arg368:NH2, Tyr116:O…Ser321:N) at this interface are preserved in the C. elegans structure, and the identities of these residues are conserved across species. In contrast, the residues forming hydrogen bonds across the second dimer interface in the human PRMT5 tetramer (Arg101:NH1…Asp531:OD1, Arg101:NH2…Asn533:O, Asn133:OD1…Trp603:NE1) are not conserved in the C. elegans sequence. The extensive and close interactions between the TIM barrel and methyltransferase domains across monomers in the human PRMT5 structure suggest that these interactions are the primary driver for oligomerization.

Structure and Interactions of MEP50.

MEP50 adopts the WD40 β-propeller structure with seven blades (Fig. 2C), six of which are composed of four β-strands, with the last β-sheet containing only three ordered strands; the first 12 and last 13 residues are disordered and not visible in the electron density. The structure therefore lacks the “velcro” closure seen in the majority of WD40 domains, where the three strands from the last blade form a β-sheet with the strand that precedes the first blade (22, 37). The sequence motifs that characterize the WD40 domain are scarce in MEP50, with only two of the seven blades containing the WD motif at the end of the third strand. One of the other blades has a WE motif, and the remaining blades have a Val, Leu, Phe, or His residue instead of Trp at this position. The Gly-His dipeptide that occurs at the end of the outer strand is absent in all seven blades of MEP50, although five of the blades contain a histidine residue preceded by a nonglycine residue at this position. The aspartic acid residue that usually occurs in the loop between the second and third strand is absent in the first blade but present in the six other blades. The top of the domain, defined as the surface formed by the loops that link the outermost strand of one blade to the innermost strand of the next blade (20), is oriented toward and interacts closely with PRMT5. This surface is the primary interaction and recognition surface for the majority of WD40 domains, although they can also interact with partner proteins through the circumference and the bottom of the barrel. The segment between β2 and β3 of the PRMT5 TIM barrel wraps around the side of the MEP50 domain, interacting closely with the outer surface of the third and fourth blade. The MEP50 molecule interacts solely with the N-terminal TIM barrel domain of PRMT5, making extensive charged and Van der Waals interactions, burying an accessible surface area of 2,027 Å2. With such a large interaction surface, and a calculated free-energy gain of 13.6 kcal/mol (38), MEP50 is very tightly bound to PRMT5.

A comparison of the MEP50 structure with structures of WDR5 (another WD40 protein and a component of many histone methyltransferase complexes) reveals distinct differences in the functional residues involved in peptide recognition in WDR5, despite a conserved overall structure. WDR5 binds both unmethylated and lysine mono-, di-, and trimethylated histone H3K4 peptides, and has recently been shown to differentiate between symmetric and asymmetric arginine di-methylation by specifically recognizing H3R2me2s (39). Peptides bind WDR5 on the top surface of the β-propeller domain, inserting the side-chain of Arg2 into the central channel where the guanidine group packs between the side-chains of Phe133 and Phe263. In the human PRMT5:MEP50 complex, the top surface of MEP50 is used for recognition of the TIM barrel of PRMT5. In addition, MEP50 lacks the arginine recognition motif of WDR5, with Ser129/Gly260 replacing the “phenylalanine clamp” (Phe133/Phe263) that binds the guanidyl side-chain in WDR5. The loop following the β2 strand of the TIM barrel domain of PRMT5 traces the top surface of MEP50 and the Arg49 side-chain interacts with Asp99 of MEP50. Because the top surface and one side of MEP50 are fully engaged in binding to PRMT5, MEP50 likely uses the bottom and the rest of the circumference of the barrel to interact with and recruit partners and substrates.

Catalytic Domain and Salient Features of the PRMT5 Cofactor Binding Site.

The catalytic domain adopts the canonical arginine methyltransferase tertiary structure similar to the type I PRMTs, with an AdoMet binding domain containing the nucleotide binding Rossmann fold, followed by a β-sandwich domain involved in substrate binding. PRMT5 lacks the YFxxY motif seen in type I PRMTs (40), and this region, which is involved in binding both cofactor and substrate, adopts a very different conformation below the AdoMet binding site in PRMT5. In addition, the conserved THW motif of the type I PRMTs is FSW in human PRMT5. Although the tryptophan residue adopts a similar conformation as the type I PRMTs, the threonine in type I PRMTs packs against the tryptophan, whereas the equivalent Phe577 of the FSW motif in PRMT5 is oriented toward the solvent, pi-stacking against Phe300 in the linker between the catalytic and TIM barrel domains.

There is clear density for the bound AdoMet analog A9145C, which binds in a similar orientation to the closely related methyltransferase inhibitor sinefungin and AdoHcy observed in type I PRMT structures (Fig. 3A). However, there are significant differences in the composition and nature of the binding interactions of the cofactor analog. In all known type I PRMT structures the adenine ring of AdoHcy makes a pair of hydrogen bonds to the protein, accepting a proton from the main-chain amino group, and donating a proton to the acidic residue that follows. In PRMT5, although the adenine ring accepts a proton from the main-chain amino of Met420, the succeeding residue is Arg421 (a basic residue), which instead stabilizes Asp419 (the residue preceding Met420), and it is Asp419 that accepts a proton from the adenine. The hydroxyl groups of the ribose moiety are stabilized by hydrogen bonds to the side-chains of Glu392 and Tyr324. The side-chain of Glu392 interacts with both hydroxyls, and is conserved in the other known PRMT structures, although it is an aspartate in PRMT3, instead of a glutamate. The Tyr324 residue in PRMT5 that hydrogen bonds to the ribose is variable among the PRMTs; it is a histidine in PRMT1 and PRMT3, and a glutamine in PRMT4. The carboxylate at the amino acid end of the ligand makes a split hydrogen bond to the side-chain of Tyr334 in PRMT5. This residue is not conserved in the other PRMTs, and is a threonine in PRMT1, PRMT3, and PRMT4. The preceding residue, Lys333, which hydrogen bonds to the carboxylate of the cofactor analog, is an arginine in all of the other PRMTs. Despite the differences in protein–ligand interactions between PRMT5 and type I PRMTs, the conformation of the AdoMet analog is similar to what has been observed in the active site of type I arginine methyltransferases.

Fig. 3.
(A) Interactions of the AdoMet analog (A9145C in black) with the PRMT5 cofactor binding site in green. The histone H4 derived substrate peptide is shown in magenta, and the omit map of A9145C is contoured at 3σ in blue. (B) A close-up view of ...

Peptide Complex and the Substrate Binding Pocket.

Our structure has clear density for the first eight residues (SGRGKGGK) of the bound peptide (Fig. 3B and Fig. S8). The histone H4-derived substrate peptide binds in a groove on the surface of the β-barrel domain, inserting the arginine side-chain (Arg3 of the peptide) through a narrow tunnel formed by Leu312, Phe327, and Trp579 to access the active site. The peptide residues that flank the arginine form a sharp β-turn at the neck of the tunnel stabilized by a hydrogen bond between the main-chain carbonyl of the Ser1 and the amino group of Gly4; the Ser1 carbonyl makes an additional hydrogen bond to the Gln309 side-chain of PRMT5. The bulk of the interactions between the substrate peptide and PRMT5 are mediated by protein backbone interactions. The main-chain carbonyls of peptide residues Gly2 and Arg3 make hydrogen bonds to the main-chain amino groups of Phe580 and Leu312, respectively, and the amino and carbonyl groups of peptide residue Lys5 makes hydrogen bonds to the carbonyl and amino groups of PRMT5 Ser310. The conformational and spatial restraints of this binding mode provide a rationale for the preference for glycine residues flanking the substrate arginine.

This structure is unique in revealing the critical role in catalysis played by the highly conserved active site glutamate residues Glu435 and Glu444 of the so-called double-E loop (41). Each of the two glutamate residues form a pair of salt bridges with the guanidine side-chain of the substrate arginine (H4R3) with the ω-NG nitrogen atom poised for methyl transfer (Fig. 3B). These glutamate residues are likely involved in de-protonating and activating the ω-NG nitrogen atom. The phenylalanine residue (Phe327) that has been shown to play a role in specifying symmetric di-methylation of PRMT5 (35) pi-stacks against the side of the guanidyl group orienting the substrate arginine for methyl-transfer. Significantly, two of the three tyrosine residues that are phosphorylated by Jak2 are involved in substrate binding. Tyr304 packs against the acetylated N-terminus of the substrate peptide, and the hydroxyl group of Tyr307 hydrogen bonds to main-chain carbonyl and amino groups of substrate residues Gly6 and Lys8, respectively.


The hetero-octameric structure of the PRMT54:MEP504 methyltransferase complex presented here likely represents the core unit that associates with different binding partners, in a context-dependent manner, to form larger multicomponent complexes that specifically methylate a diverse set of substrates in both the cytoplasm and the nucleus. The interactions between the PRMT5 monomers in the complex are very different from the conserved dimer interaction observed in the structures of type I PRMTs. In the type I PRMT dimers, the “dimerization arm” from the β-barrel domain of each monomer interacts with the AdoMet binding domain of the second monomer in a head-to-tail orientation, and this interaction has been shown to be necessary for cofactor binding and activity (41, 42). In contrast, the corresponding regions in human PRMT5 interact directly with each other at the center of the oligomer. Apart from this interaction between catalytic domain residues, the bulk of the interactions between the PRMT5 monomers are mediated by the N-terminal TIM barrel domains. The PRMT5 TIM barrel domain therefore plays dual structural roles, promoting oligomerization by interacting with the catalytic domain to form the PRMT5 tetramer, and binding specifically and tightly to MEP50 molecules.

TIM barrel domains, composed of eight β/α-segments that fold to form an internal β-barrel surrounded by helices, are one of the most well-studied structural families (43). The low sequence similarity among TIM barrel proteins coupled with the functional diversity of this structural class has led to extensive research on the potential role of convergent and divergent evolution in generating the ability to catalyze various reactions using a conserved structural scaffold. TIM barrel proteins are typically enzymes involved in molecular or energy metabolism, and additional studies are warranted to elucidate whether the PRMT5 TIM barrel domain has any catalytic activity. In TIM barrel enzymes, the loops linking the C-terminal ends of the β-strands to the helical segments contribute the residues that are involved in catalysis, metal-ligation, and phosphate-binding. The equivalent site of the PRMT5 TIM barrel is oriented away from the methyltransferase catalytic domain and interacts intimately with the MEP50 molecule.

Comparison of the human PRMT5 monomer to the dimeric C. elegans structure suggests that the C. elegans monomer has possibly been misinterpreted as being composed of the equivalent of the TIM barrel domain from one polypeptide chain and the catalytic domain from another chain (Fig. S7). This misinterpretation may have been precipitated by the fact that the region between the TIM barrel and the catalytic domain is disordered in the C. elegans structure. In the human structure there is no ambiguity, because the equivalent segment between the TIM barrel and catalytic domain (residues 293–320) is fully ordered. The preservation of residues and interactions across this dimer interface between the human and C. elegans structures suggests that this dimer is a conserved quaternary structure of PRMT5 across species, and likely to be the structure adopted by human PRMT5 in the absence of MEP50. In contrast, the residues at the second dimer-interaction surface in the human PRMT5:MEP50 structure are not conserved across species, and the formation of the tetramer is brought about by the binding of MEP50. A sequence search did not identify a MEP50 ortholog in C. elegans, suggesting that the nematode PRMT5 may have a different mechanism of recruiting binding partners and substrates. Contrary to expectation, the structure of the human PRMT5:MEP50 complex revealed that MEP50 does not bind at any of the PRMT5 oligomerization interfaces but binds on the outer surface of the tetrameric PRMT5 core, interacting solely with the TIM barrel domain. Therefore, the binding of MEP50 must introduce a subtle conformational change that favors the formation of PRMT5 tetramers, and also lowers the Km for AdoMet and substrate peptide.

The observation from the structure that two of the tyrosine residues (Tyr304 and Tyr307) that are phosphorylated by oncogenic V617F Jak2 (17) to down-regulate the methyltransferase activity are involved in substrate binding, provides a rationale for the observed loss of activity upon phosphorylation, because phosphorylation would disrupt the substrate pocket. The occurrence of these residues on a short helix and loop that is part of the linker between the TIM barrel and catalytic domains suggests a possible regulatory role for this segment.

The observed lack of processivity in successive mono- and di-methylation by PRMT5 is due to the requirement of the monomethylated substrate having to dissociate from the enzyme and bind again with the ω-N′G nitrogen atom poised for methylation. The partial processivity of type I PRMTs, such as PRMT1, is likely enabled by its retention of the monomethylated product in the active site, while allowing the methyl group on the arginine to rotate about the Cζ-ωNG bond and re-present the methylated nitrogen atom for the second methylation in concert with the replacement of AdoHcy by AdoMet in the cofactor binding site. In type II PRMTs, retention of the peptide in the active site for processive di-methylation would require larger rotations of side-chain torsion angles to interchange the positions of the ω nitrogen atoms, which would be precluded by the spatial constraints of the active site. In the cellular environment a monomethylated histone tail may remain in the vicinity for successive methylation, because the octameric histone complex with multiple substrates (H2A, H3, and H4) could simultaneously bind multiple active sites in the PRMT54:MEP504 complex. The documented ability of the PRMT5 F327M mutant to perform both symmetric and asymmetric di-methylation (35) is likely enabled by its capacity to accommodate the methyl group of monomethylated substrate and still present the appropriate nitrogen for both symmetric or asymmetric di-methylation. The conformational flexibility of the methionine side-chain potentially allows sufficient compliance in binding monomethylated arginine, so that it can present the appropriate terminal ω-NG nitrogen atom toward AdoMet.

The extensive and tight interaction between MEP50 and PRMT5 is consistent with the role of MEP50 as the primary binding partner of PRMT5, and is also evidenced by the presence of MEP50 in several of the characterized PRMT5 complexes (Fig. S1). The increased stability and methyltransferase activity of the hetero-octameric PRMT5:MEP50 complex compared with isolated PRMT5 lends further credence to the view that the PRMT5:MEP50 complex is the functional biological module. The well-documented interaction of MEP50 with many of the binding partners and substrates of PRMT5 is suggestive of an additional role for MEP50 in recruiting substrates and partners to the methyltransferase, and reflective of the broader role of WD40 proteins in epigenetic pathways.

The view revealed by this structure clearly delineates the interactions between the methyltransferase and the substrate peptide, and clarifies the roles of the active site residues in catalysis. Thus, in addition to advancing our understanding of this complex target, this work also provides a valuable resource for structure based drug design efforts on this emerging target class.


Full-length PRMT5 (NP_006100, residues 1–637) and MEP50 (NP_077007, residues 2–342) were coexpressed in insect cells and PRMT5:MEP50 complex purified by affinity followed by size-exclusion chromatography. Crystals were grown at 8 °C in sitting drops with protein incubated with peptide and A9145C. Detailed methods are provided in SI Methods and data and refinement statistics are included in Table S1.

Supplementary Material

Supporting Information:


We thank Guemalli Cardona for work on the enzyme inhibition measurements. Use of the Advanced Photon Source at Argonne National Laboratory was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357.


Conflict of interest statement: This research was funded by Lilly Research Laboratories and all the authors are employees of Eli Lilly and Company.

*This Direct Submission article had a prearranged editor.

Data deposition: The atomic coordinates and structure factors have been deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank, www.rscb.org (PDB ID code 4GQB).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1209814109/-/DCSupplemental.


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