Biochemical Investigation of the Interaction of Picln, Riok1 and COPR5 with the PRMT5–MEP50 Complex

Communications ChemBioChem doi.org/10.1002/cbic.202100079 1 2 3 Biochemical Investigation of the Interaction of pICln, RioK1 4 5 and COPR5 with the PRMT5–MEP50 Complex 6 [a, b] [c] [d, e] ' [f] 7 Adrian Krzyzanowski, Raphael Gasper, Hélène Adihou, Peter t Hart,* and [a, b] 8 Herbert Waldmann* 9 10 compete for the same binding site, but are not competitive 11 The PRMT5–MEP50 methyltransferase complex plays a key role with MEP50.[5] 12 in various cancers and is regulated by different protein–protein PRMT5 contains three domains, that is, the catalytic site- 13 interactions. Several proteins have been reported to act as containing Rossmann fold, a β-barrel domain used for dimeriza- 14 adaptor proteins that recruit substrate proteins to the active tion, and the TIM barrel, which acts as interaction site for 15 site of PRMT5 for the methylation of arginine residues. To MEP50 (Figure 1A).[3a,c] PRMT5 and MEP50 form a hetero- 16 define the interaction between these adaptor proteins and octameric complex whose structure has been determined,[3a] 17 PRMT5, we employed peptide truncation and mutation studies and structural information on the interaction between PRMT5 18 and prepared truncated protein constructs. We report the and the other adaptor proteins has only very recently been 19 characterisation of the interface between the TIM barrel of described in preliminary form.[7] Overexpression of PRMT5 is 20 PRMT5 and the adaptor proteins pICln, RioK1 and COPR5, and frequently observed in cancer which makes the protein an 21 identify the consensus amino acid sequence GQF[D/E]DA[E/D] intensively investigated anticancer target, and active-site di- 22 involved in binding. Protein crystallography revealed that the rected inhibitors have reached clinical trials.[8] Further study of 23 RioK1 derived peptide interacts with a novel PPI site. the PRMT5 PPIs and binding partners is important for a better 24 understanding of its regulation. 25 Herein, we describe the use of synthetic peptides to study 26 Introduction the PRMT5–adaptor protein interaction by biochemical meth- 27 ods and protein crystallography. An adaptor protein consensus 28 Methylation of terminal arginine nitrogens by protein arginine sequence was derived by sequence alignment and synthetic 29 methyltransferases (PRMTs) plays important roles in various peptides derived from this consensus were prepared. The site 30 cellular processes, including the establishment of cancer.[1] of interaction was initially determined by truncations of PRMT5 31 Symmetric methylation of both arginine nitrogens is mainly itself and measuring the affinity for synthetic adaptor protein 32 catalysed by PRMT5, which methylates histones H2A, H3 and H4 peptides. Truncation and alanine scanning of the adaptor 33 and various non-histone targets.[2] Target selection depends on protein peptides allowed identification of the residues contribu- 34 adaptor proteins which bind to PRMT5 and recruit specific ting to the interaction. A co-crystal structure of a RioK1 derived 35 substrates to the PRMT5 active site.[2a] Adaptor proteins include peptide with the PRMT5 TIM barrel domain confirmed the 36 MEP50 (WDR77; which plays an important role in histone results of our mutation and truncation studies. Our findings are 37 methylation),[3] pICln (which recruits Sm proteins),[4] RioK1 in agreement with the recent preliminary report by Sellers 38 (which recruits nucleolin)[5] and COPR5 (which recruits histone et al.[7] 39 H4).[6] pICln and RioK1 bind mutually exclusive to PRMT5 and 40 41 42 43 44 [a] A. Krzyzanowski, Prof. Dr. H. Waldmann [e] Dr. H. Adihou Department of Chemical Biology Medicinal Chemistry 45 Max Planck Institute of Molecular Physiology Research and Early Development Cardiovascular, Renal and Metabolism, 46 Otto-Hahn-Strasse 11, 44227 Dortmund (Germany) BioPharmaceuticals R&D 47 E-mail: [email protected] AstraZeneca [b] A. Krzyzanowski, Prof. Dr. H. Waldmann Otto-Hahn-Strasse 11, 44227 Gothenburg (Sweden) 48 Faculty of Chemistry, Chemical Biology [f] Dr. Peter 't Hart 49 Technical University Dortmund Chemical Genomics Centre of the Max Planck Society 50 Otto-Hahn-Strasse 6, 44221 Dortmund (Germany) Max Planck Institute of Molecular Physiology [c] Dr. R. Gasper Otto-Hahn-Strasse 11, 44227 Dortmund (Germany) 51 Crystallography and Biophysics Unit E-mail: [email protected] 52 Max-Planck-Institute of Molecular Physiology Supporting information for this article is available on the WWW under 53 Otto-Hahn-Strasse 11, 44227 Dortmund (Germany) https://doi.org/10.1002/cbic.202100079 [d] Dr. H. Adihou © 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH. This is 54 AstraZeneca MPI Satellite Unit an open access article under the terms of the Creative Commons Attribution 55 Department of Chemical Biology License, which permits use, distribution and reproduction in any medium, Max Planck Institute of Molecular Physiology 56 provided the original work is properly cited. Otto-Hahn-Strasse 11, 44227 Dortmund (Germany) 57 ChemBioChem 2021, 22, 1–8 1 © 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH These are not the final page numbers! �� Wiley VCH Mittwoch, 31.03.2021 2199 / 198936 [S. 1/8] 1 Communications ChemBioChem doi.org/10.1002/cbic.202100079 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Figure 1. Interaction between PRMT5 and its adaptor proteins. A) Schematic representation of the PRMT5–adaptor protein complexes with their methylation 44 substrate. The PRMT5–MEP50 complex was visualised based on the PDB structure 4GQB. B) BLAST alignments for pICln, RioK1, COPR5 and DAB1. C) Results of 45 the fluorescence polarization measurements for interaction of the identified peptide sequences with the full PRMT5–MEP50 complex, truncated TIM–MEP50 and isolated TIM barrel domain (n=3). D) Results of fluorescence polarisation assays for histone tail peptides and the truncated protein complex TIM–MEP50 46 (n=3). 47 48 49 Results and Discussion comparison of the protein sequences. Protein BLAST alignment 50 identified significant similarity in sequences between the C- 51 Intrigued by the report of Guderian et al., noting mutually terminal region of pICln (TPTVAGQFEDAD, residues 223–234) 52 exclusive binding of pICln and RioK1 to PRMT5, we aimed to and the N-terminal region of RioK1 (SRVVPGQFDDAD, residues 53 identify common features between the two proteins to identify 9–20) as shown in Figure 1B.[9] The identified sequences were 54 a potential interaction domain.[5] Due to the very limited then used for a global similarity search among available Homo 55 structural information available for pICln and RioK1, at the time sapiens protein data, resulting in the identification of two new 56 of initiating the project, we focused on the analysis and 57 ChemBioChem 2021, 22, 1–8 www.chembiochem.org 2 © 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH These are not the final page numbers! �� Wiley VCH Mittwoch, 31.03.2021 2199 / 198936 [S. 2/8] 1 Communications ChemBioChem doi.org/10.1002/cbic.202100079 sequence matches: COPR5 (GQFDDAE, residues 177–183) and 1.14 μM) and 3 (K 0.55 μM), but not 4 (Figure 1C top left and 1 D DAB1 (PTVAGQF, residues 388–394; Figure 1B). Table 1). 2 Notably, the identified sequences of RioK1, pICln and In order to identify the protein domain responsible for the 3 COPR5 were part of the protein regions, previously proposed to interaction with the peptides, a truncated PRMT5 protein 4 be responsible for binding to PRMT5 (Figure 1C). The C-terminal (residues 1–292), corresponding to the TIM barrel domain, was 5 region of pICln containing the acid domain AD3 (residues 230– either expressed alone or co-expressed as a complex with 6 237), and regions in the PH domain (residues 1–134) were MEP50 (Figure 1C). The truncation constructs lack key residues 7 shown to mediate the PPI with PRMT5.[4b,10] Moreover, the N- required for higher order oligomer formation and are therefore 8 terminal fragment of RioK1 (residues 1–120) and the C-terminal expected to exist as monomers which was confirmed by mass 9 fragment of COPR5 (residues 141–184) were previously reported photometry (Figure S8).[3a,11] 10 as the main contributors to the interactions with the meth- FP analysis of compounds 1–3 gave similar K values for all 11 D yltransferase.[5,6] We noted that in all cases the common GQF[D/ three tested protein constructs (Figure 1C and Table 1). These 12 E]DA[E/D] sequence occurred in the regions involved in the PPIs results clearly indicate that the peptides interact with the TIM 13 with PRMT5. The same sequences were identified by Sellers barrel domain of PRMT5. The lower binding affinity to the 14 et al. by applying BioGrid analysis.[9] isolated TIM barrel domain in comparison with the complexes 15 Fluorescently labelled peptides, 1, 2, 3 and 4 (Table 1), containing MEP50, may be due to a potential loss of the 16 corresponding to the determined sequence matches for RioK1, stabilising effect of MEP50 on PRMT5. The labelled peptides did 17 pICln, COPR5 and DAB1, respectively, were synthesised to be not show unspecific binding to BSA and GST (Figure S3 and S4). 18 tested for binding by using a fluorescence polarisation (FP) Compound 4 did not have affinity for any of the protein 19 assay. An untagged, native, human PRMT5–MEP50 complex was constructs indicating the negatively charged C-terminal section 20 expressed and tested for activity using the MTAse Glo assay is critical for binding. 21 (Figure S10 in the Supporting Information). When tested for In order to further define the requirements for pICln/RioK1/ 22 binding we observed potent interaction of 1 (K 0.52 μM), 2 (K COPR5-PRMT5 binding, we synthesised a series of RioK1 and 23 D D pICln derived peptides (Table 2) to determine the shortest 24 25 26 Table 1.

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