Structure of the Arginine Methyltransferase PRMT5-MEP50 Reveals a Mechanism for Substrate Specificity

et al. (2013) Structure of the Arginine Methyltransferase PRMT5-MEP50 Reveals a Mechanism for Substrate Specificity. PLoS ONE 8(2): e57008. doi:10.1371/journal.pone.0057008 Structure of the Arginine Methyltransferase PRMT5- MEP50 Reveals a Mechanism for Substrate Specificity Meng-Chiao Ho 0 Carola Wilczek 0 Jeffrey B. Bonanno 0 Li Xing 0 Janina Seznec 0 Tsutomu Matsui 0 Lester G. Carter 0 Takashi Onikubo 0 P. Rajesh Kumar 0 Man K. Chan 0 Michael Brenowitz 0 R. Holland Cheng 0 Ulf Reimer 0 Steven C. Almo 0 David Shechter 0 Sue Cotterill, St. Georges University of London, United Kingdom 0 1 Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University , Bronx , New York, United States of America, 2 Department of Physiology and Biophysics, Albert Einstein College of Medicine of Yeshiva University , Bronx , New York, United States of America, 3 Institute of Biological Chemistry , Academia Sinica, Nankang, Taipei, Taiwan, 4 JPT Peptide Technologies, Berlin, Germany , 5 Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory , Menlo Park , California, United States of America, 6 Department of Molecular and Cellular Biology, University of California Davis , Davis, California , United States of America The arginine methyltransferase PRMT5-MEP50 is required for embryogenesis and is misregulated in many cancers. PRMT5 targets a wide variety of substrates, including histone proteins involved in specifying an epigenetic code. However, the mechanism by which PRMT5 utilizes MEP50 to discriminate substrates and to specifically methylate target arginines is unclear. To test a model in which MEP50 is critical for substrate recognition and orientation, we determined the crystal structure of Xenopus laevis PRMT5-MEP50 complexed with S-adenosylhomocysteine (SAH). PRMT5-MEP50 forms an unusual tetramer of heterodimers with substantial surface negative charge. MEP50 is required for PRMT5-catalyzed histone H2A and H4 methyltransferase activity and binds substrates independently. The PRMT5 catalytic site is oriented towards the crossdimer paired MEP50. Histone peptide arrays and solution assays demonstrate that PRMT5-MEP50 activity is inhibited by substrate phosphorylation and enhanced by substrate acetylation. Electron microscopy and reconstruction showed substrate centered on MEP50. These data support a mechanism in which MEP50 binds substrate and stimulates PRMT5 activity modulated by substrate post-translational modifications. - Funding: Data for this study were measured at beamline X25 and X29A of the National Synchrotron Light Source. Financial support comes principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy, and from the National Center for Research Resources (NCRR, P41RR012408) and the National Institute of General Medical Sciences (P41GM103473) of the National Institutes of Health (NIH). Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH, NCRR, Biomedical Technology Program (P41RR001209). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH. This research was partially supported by grants from Academia Sinica, Taiwan, and from the National Science Council, Taiwan (NSC101-2311-B-001-002) to MHC, the NIH (GM093342 to SCA and AI095382 to RHC) and the Albert Einstein Cancer Center (CA013330). DS is a Scholar of the Alexander and Alexandrine Sinsheimer Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have the following interests: UR and JS are employees of JPT Peptide Technologies. JPT has developed, manufactures, and distributes peptide microarrays similar to those used in this study. The arrays used in the study were developed further to a marketed product (His-MA_01) available at JPT. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. The peptide array used in this study has been developed and manufactured by JPT. This library on this array has been extended and is marketed as Histone Code Peptide Microarray (His-MA_01) by JPT. . These authors contributed equally to this work. The family of protein arginine methyltransferases (PRMTs) in metazoans includes at least 10 proteins with diverse roles [1]. The majority of these enzymes are Type I enzymes that are capable of mono- and asymmetric dimethylation of arginine, with Sadenosylmethionine (SAM) as the methyl donor. PRMT5 is a Type II enzyme, capable of mono- and symmetric dimethylation [24]. PRMT5 methylates histones H2A and H4 on Arg3 [5], histone H3 on Arg2 [6] and Arg8, and many other proteins [7]. PRMT5 is required for stem cell maintenance and developmental growth in Planaria [8], in mouse embryonic and induced pluripotent stem cells [9,10], and is required for initiation of differentiation in myogenesis [11]. PRMT5 prevents keratinocyte differentiation [12] and may be responsible for stem cell maintenance in germ cell tumors [13]. PRMTs and histone arginine methylation are heavily enriched in eggs and early embryos of metazoans [5,9,14]. We previously showed that PRMT5-MEP50 methylates histones H2A and H4 and the histone chaperone Nucleoplasmin in Xenopus laevis eggs [5]. Furthermore, PRMT5 regulates transcription via histone methylation, specifically down-regulating transcription of ribosomal genes, cyclin E, Rb, and other genes [1517]. PRMT5 partners with many protein cofactors, including Blimp1 [14], RioK1 [18], pICLn [19], MBD/NuRD [20], and MEP50 [21]. MEP50, a WD-40 repeat protein, is its most common partner and likely present in every PRMT5-containing complex in vivo [1]. Recent reports demonstrated that phosphorylation of PRMT5 by mutant Jak2 kinase and of MEP50 by Cdk4 altered the activity and targeting of the PRMT5 enzyme leading to tumorigenesis [22,23]. Insight into the location of these phosphorylation sites would illuminate the potential oncogenic mechanisms promoted by these aberrant kinase targets. Furthermore, how PRMT5 interacts with protein cofactors to alter its activity and gain substrate specificity is unclear. PRMT5 forms high molecular weight complexes [24]. X. laevis PRMT5-MEP50 (XlPRMT5-MEP50) forms an assembly larger than expected for a simple heterodimer pair [5]. PRMT1, PRMT3 and PRMT4 (CARM1) dimerize using a dimerization arm located at the C-terminus [25]. The structure of C. elegans PRMT5 (CePRMT5) exhibited a head-to-tail dimer, with the Nterminus of one PRMT (...truncated)