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.epigenetics | protein-protein complex | A9145C P osttranslational 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 (1-3). 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-repe...
The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.
Loss-of-function mutations in the retinoblastoma gene RB1 are common in several treatment-refractory cancers such as small-cell lung cancer and triplenegative breast cancer. To identify drugs synthetic lethal with RB1 mutation (RB1 mut), we tested 36 cell-cycle inhibitors using a cancer cell panel profi ling approach optimized to discern cytotoxic from cytostatic effects. Inhibitors of the Aurora kinases AURKA and AURKB showed the strongest RB1 association in this assay. LY3295668, an AURKA inhibitor with over 1,000-fold selectivity versus AURKB, is distinguished by minimal toxicity to bone marrow cells at concentrations active against RB1 mut cancer cells and leads to durable regression of RB1 mut tumor xenografts at exposures that are well tolerated in rodents. Genetic suppression screens identifi ed enforcers of the spindle-assembly checkpoint (SAC) as essential for LY3295668 cytotoxicity in RB1-defi cient cancers and suggest a model in which a primed SAC creates a unique dependency on AURKA for mitotic exit and survival. SIGNIFICANCE: The identifi cation of a synthetic lethal interaction between RB1 and AURKA inhibition, and the discovery of a drug that can be dosed continuously to achieve uninterrupted inhibition of AURKA kinase activity without myelosuppression, suggest a new approach for the treatment of RB1-defi cient malignancies, including patients progressing on CDK4/6 inhibitors.
Background: SMYD2 is a methyltransferase whose role in cancer is poorly understood and is lacking cell-active chemical tools.Results: We describe LLY-507, a small molecule inhibitor of SMYD2.Conclusion: LLY-507 is potent, selective, cell-active, and binds SMYD2 in a high resolution co-crystal.Significance: LLY-507 is a first-in-class cell-potent chemical probe that will be valuable in dissecting SMYD2 biology.
Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyzes the formation of symmetric dimethylarginine in a number of nuclear and cytoplasmic proteins. Although the cellular functions of PRMT5 have not been fully unraveled, it has been implicated in a number of cellular processes like RNA processing, signal transduction, and transcriptional regulation. PRMT5 is ubiquitously expressed in most tissues and its expression has been shown to be elevated in several cancers including breast cancer, gastric cancer, glioblastoma, and lymphoma. Here, we describe the identification and characterization of a novel and selective PRMT5 inhibitor with potent and activity. Compound (also called LLY-283) inhibited PRMT5 enzymatic activity and in cells with IC of 22 ± 3 and 25 ± 1 nM, respectively, while its diastereomer, compound (also called LLY-284), was much less active. Compound also showed antitumor activity in mouse xenografts when dosed orally and can serve as an excellent probe molecule for understanding the biological function of PRMT5 in normal and cancer cells.
The enhancer-of-zeste homolog 2 (EZH2) gene product is an 87 kDa polycomb group (PcG) protein containing a C-terminal methyltransferase SET domain. EZH2, along with binding partners, i.e., EED and SUZ12, upon which it is dependent for activity forms the core of the polycomb repressive complex 2 (PRC2). PRC2 regulates gene silencing by catalyzing the methylation of histone H3 at lysine 27. Both overexpression and mutation of EZH2 are associated with the incidence and aggressiveness of various cancers. The novel crystal structure of the SET domain was determined in order to understand disease-associated EZH2 mutations and derive an explanation for its inactivity independent of complex formation. The 2.00 Å crystal structure reveals that, in its uncomplexed form, the EZH2 C-terminus folds back into the active site blocking engagement with substrate. Furthermore, the S-adenosyl-L-methionine (SAM) binding pocket observed in the crystal structure of homologous SET domains is notably absent. This suggests that a conformational change in the EZH2 SET domain, dependent upon complex formation, must take place for cofactor and substrate binding activities to be recapitulated. In addition, the data provide a structural context for clinically significant mutations found in the EZH2 SET domain.
Microsomal prostaglandin E synthase 1 (mPGES-1) is an α-helical homotrimeric integral membrane inducible enzyme that catalyzes the formation of prostaglandin E2 (PGE2) from prostaglandin H2 (PGH2). Inhibition of mPGES-1 has been proposed as a therapeutic strategy for the treatment of pain, inflammation, and some cancers. Interest in mPGES-1 inhibition can, in part, be attributed to the potential circumvention of cardiovascular risks associated with anti-inflammatory cyclooxygenase 2 inhibitors (coxibs) by targeting the prostaglandin pathway downstream of PGH2 synthesis and avoiding suppression of antithrombotic prostacyclin production. We determined the crystal structure of mPGES-1 bound to four potent inhibitors in order to understand their structure-activity relationships and provide a framework for the rational design of improved molecules. In addition, we developed a light-scattering-based thermal stability assay to identify molecules for crystallographic studies.
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