The structure-activity relationships of L3MBTL3 inhibitors: flexibility of the dimer interface.
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ABSTRACT:
SUBMITTER: Camerino MA
PROVIDER: S-EPMC3897169 | BioStudies | 2013-01-01
REPOSITORIES: biostudies
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Project description:We describe the discovery of UNC1215, a potent and selective chemical probe for the methyllysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin-interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a K(d) of 120 nM, competitively displacing mono- or dimethyllysine-containing peptides, and is greater than 50-fold more potent toward L3MBTL3 than other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a unique 2:2 polyvalent mode of interaction between UNC1215 and L3MBTL3. In cells, UNC1215 is nontoxic and directly binds L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins, and point mutants that disrupt the Kme-binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215 on localization. Finally, UNC1215 was used to reveal a new Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.
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Project description:Lysine methylation is a key epigenetic mark, the dysregulation of which is linked to many diseases. Small-molecule antagonism of methyl-lysine (Kme) binding proteins that recognize such epigenetic marks can improve our understanding of these regulatory mechanisms and potentially validate Kme binding proteins as drug-discovery targets. We previously reported the discovery of 1 (UNC1215), the first potent and selective small-molecule chemical probe of a methyl-lysine reader protein, L3MBTL3, which antagonizes the mono- and dimethyl-lysine reading function of L3MBTL3. The design, synthesis, and structure-activity relationship studies that led to the discovery of 1 are described herein. These efforts established the requirements for potent L3MBTL3 binding and enabled the design of novel antagonists, such as compound 2 (UNC1679), that maintain in vitro and cellular potency with improved selectivity against other MBT-containing proteins. The antagonists described were also found to effectively interact with unlabeled endogenous L3MBTL3 in cells.
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Project description:L3MBTL3 recognizes mono- and dimethylated lysine residues on histone tails. The recently reported X-ray cocrystal structures of the chemical probe UNC1215 and inhibitor UNC2533 bound to the methyl-lysine reading MBT domains of L3MBTL3 demonstrate a unique and flexible 2:2 dimer mode of recognition. In this study, we describe our in vitro analysis of L3MBTL3 dimerization via its MBT domains and additionally show that this dimerization occurs within a cellular context in the absence of small molecule ligands. Furthermore, mutations to the first and second MBT domains abrogated L3MBTL3 dimerization both in vitro and in cells. These observations are consistent with the hypothesis that L3MBTL3 engages methylated histone tails as a dimer while carrying out its normal function and provides an explanation for the presence of repeated MBT domains within L3MBTL3.
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Project description:The Polycomb group (PcG) of proteins is a family of important developmental regulators. The respective members function as large protein complexes involved in establishment and maintenance of transcriptional repression of developmental control genes. MBTD1, Malignant Brain Tumor domain-containing protein 1, is one such PcG protein. MBTD1 contains four MBT repeats.We have determined the crystal structure of MBTD1 (residues 130-566aa covering the 4 MBT repeats) at 2.5 A resolution by X-ray crystallography. The crystal structure of MBTD1 reveals its similarity to another four-MBT-repeat protein L3MBTL2, which binds lower methylated lysine histones. Fluorescence polarization experiments confirmed that MBTD1 preferentially binds mono- and di-methyllysine histone peptides, like L3MBTL1 and L3MBTL2. All known MBT-peptide complex structures characterized to date do not exhibit strong histone peptide sequence selectivity, and use a "cavity insertion recognition mode" to recognize the methylated lysine with the deeply buried methyl-lysine forming extensive interactions with the protein while the peptide residues flanking methyl-lysine forming very few contacts [1]. Nevertheless, our mutagenesis data based on L3MBTL1 suggested that the histone peptides could not bind to MBT repeats in any orientation.The four MBT repeats in MBTD1 exhibits an asymmetric rhomboid architecture. Like other MBT repeat proteins characterized so far, MBTD1 binds mono- or dimethylated lysine histones through one of its four MBT repeats utilizing a semi-aromatic cage.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.
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