CE11 - Caractérisation des structures et relations structure-fonction des macromolécules biologiques

STRUCTURAL INSIGHTS INTO PRMT COMPLEXES: DECIPHERIING REGULATION OF ARGININE METHYLATION BY PROTEIN ARGININE METHYLTRANSFERASES. – InSiPRMTs

InSiPRMTs

Deciphering regulation of arginine methylation by Protein Arginine methyltransferases.

STRUCTURAL INSIGHTS INTO PRMT COMPLEXES

InSiPRMTs is a project of basic research intended to address several fundamental aspects of molecular recognition involving PRMTs by providing new clues for three challenges that remain to be solved by structural biology: (i) understanding how PRMTs recognize and bind their full-length substrates; (ii) revealing how full-length PRMTs achieve specific arginine methylation on different target sites; (iii) and characterizing at the molecular level functional macromolecular complexes involving PRMT targets. The aims are to provide new routes to understand PRMT substrate recognition specificity, selectivity and molecular mechanism, and more generally, deciphering regulation of arginine methylation. Based on the strong background of the partners of the consortium, it proposes to develop innovative tools to reveal snapshots of methylation complexes in productive states.<br />The central idea of the project is to produce and structurally characterize PRMT-full length substrate complexes using a modified arginine (methylation target site) as tethering tool to freeze a functional state of PRMT complexes just prior to the methyl transfer. The aims are to give structural information on different functional methylation complexes, revealing overall positions of all partners, short range and long-range interactions crucial for the methylation reaction. Methods and protocols developed for this project may be then extended to other PRMT complexes.

Full-length PRMT/partners complexes will be produced and characterized by several biophysical methods (DLS, AUC,SEC/MALS, SAXS). Mapping of PRMT substrate interactions is done by Native MS in combination with crosslinking and HDX-MS. 3D-structures determination is done using X-ray crystallography and Single Particle Cryo-EM.

The results are confidential until published in international journals. Results will not be presented here.

Arginine methylation is now considered to be a far more prevalent and important PTM than previously thought. Although once thought to be confined to histones and RNA binding proteins, it is now understood that arginine methylation targets are much more diverse. Altogether, these methylation events impact every aspect of cellular biology and human health. It is therefore not surprising that dysfunction of arginine methylation is correlated to the development of several diseases. PRMTs are modular proteins usually active as homodimers with each monomer containing a common catalytic methyltransferase domain to which additional domains may be added. PRMT7 (and probably PRMT9) are monomers which mimic the classical PRMT dimer. The presence of additional domains can strongly affect enzymatic activity, substrate binding, regulation confering structural and functional specificity to each PRMT. Additionnal domains are quite often predicted to be partly disordered, or behave as wobbly domains. Despite extensive research by many teams, known PRMT structures are mainly restricted to the catalytic domain, no structures have been solved for PRMTs in complex with full-length target proteins. To perform their function, PRMTs are usually part of larger functional complexes that include other modulator/adaptor proteins, and little is known about how proteins function dynamically within these macromolecular assemblies. PRMT-based biological processes probably require interplay and structural re-arrangements involving the catalytic module and additional modules within a given PRMT.

In preparation

Protein arginine methylation is a widespread and abundant post-translational modification (PTM) that has emerged as a major mechanism for regulating protein function in eukaryotic cells. Arginine methylation is now considered to be a far more prevalent and important post-translational modification than previously thought. Although once thought to be confined to histones and RNA binding proteins, it is now understood that arginine methylation targets are much more diverse. Altogether, these methylation events impact every aspect of cellular biology and human health. It is therefore not surprising that dysfunction of arginine methylation is correlated to the development of several diseases. Protein arginine methylation is catalyzed by protein arginine N-methyltransferases (PRMTs). Mammalian PRMTs are classified into three main types based on their methylation activity: type I PRMTs (PRMT1, PRMT2, PRMT3, PRMT4/CARM1, PRMT6 and PRMT8) dimethylate arginine asymmetrically (ADMA); type II PRMTs (PRMT5, PRMT9) dimethylate arginine symmetrically (SDMA); and type III PRMTs (PRMT7) only monomethylate arginine (MMA). PRMTs are usually active as homodimers with each monomer containing a common catalytic methyltransferase domain to which additional domains may be added. PRMT7 and PRMT9 are monomer which mimics the classical PRMT dimer. The presence of additional domains can strongly affect enzymatic activity, substrate binding and regulation thus confering structural and functional specificity to each PRMT. Additionnal domains are quite often predicted to be partly disordered, or behave as wobbly domains. Despite extensive research by many teams, known PRMT structures are mainly restricted to the catalytic domain, no structures have been solved for PRMTs in complex with full-length target proteins.

A major challenge in the epigenetic field is the identification, characterization and structural analysis of functional complexes involving epigenetic players. Structural visions of such complexes are required to understand how proteins function dynamically within a larger macromolecular assembly. Many of those complexes are transient assemblies and therefore their studies at the structural level represent one of the big challenges of today’s structural biology. The InSiPRMTs consortium will address this challenge by providing useful tools to capture and characterize complexes involving PRMTs and therefore to understand PRMTs substrate recognition specificity, selectivity and molecular mechanism. More generally, InSiPRMTs aims to decipher the regulation of arginine methylation at the atomic scale. As covalent tethering of substrates to enzymes or cofactors has proven to be an effective strategy for capturing enzyme-substrate complexes and enabling their structural characterization, InSiPRMTs propose new routes to stabilize complexes involving a given PRMT and its protein substrates.

The multidisciplinary approach developed by 3 academic research teams of complementary expertise makes relevant the choice of the “Projet de recherche collaborative” funding instrument. The success of the InSiPRMTs project indeed strongly relies on the expertise in several complementary fields: organic synthesis and chemical biology (partner 2), structural biology (partner 1), mass spectrometry (partner 3). Partners 1 and 2 have been working together in the PRMT field since several years. Both have tight collaborations with partner 3. Mass spectrometry (partner 3) will indeed be key to characterize the compounds synthesized in each task.

Project coordination

Jean Cavarelli (Institut de Génétique et de Biologie Moléculaire et Cellulaire)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IPHC Institut Pluridisciplinaire Hubert Curien - IPHC (UMR 7178)
CAMB_UNISTRA Laboratoire de Conception et Application de Molécules Bioactives (UMR 7199)
IGBMC Institut de Génétique et de Biologie Moléculaire et Cellulaire

Help of the ANR 550,918 euros
Beginning and duration of the scientific project: September 2019 - 42 Months

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