Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain.
ABSTRACT: 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.
Project description:We recently reported the discovery of UNC1215, a potent and selective chemical probe for the L3MBTL3 methyllysine reader domain. In this article, we describe the development of structure-activity relationships (SAR) of a second series of potent L3MBTL3 antagonists which evolved from the structure of the chemical probe UNC1215. These compounds are selective for L3MBTL3 against a panel of methyllysine reader proteins, particularly the related MBT family proteins, L3MBTL1 and MBTD1. A co-crystal structure of L3MBTL3 and one of the most potent compounds suggests that the L3MBTL3 dimer rotates about the dimer interface to accommodate ligand binding.
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.
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.
Project description:Methyllysine histone code readers constitute a new promising group of potential drug targets. For instance, L3MBTL1, a malignant brain tumor (MBT) protein, selectively binds mono- and di-methyllysine epigenetic marks (KMe, KMe(2) ) that eventually results in the negative regulation of multiple genes through the E2F/Rb oncogenic pathway. There is a pressing need in potent and selective small-molecule probes that would enable further target validation and might become therapeutic leads. Such an endeavor would require efficient tools to assess the free energy of protein-ligand binding. However, due to an unparalleled function of the MBT binding pocket (i.e., selective binding to KMe/KMe(2) ) and because of its distinctive structure representing a small aromatic "cage," an accurate assessment of its binding affinity to a ligand appears to be a challenging task. Here, we report a comparative analysis of computationally affordable affinity predictors applied to a set of seven small-molecule ligands interacting with L3MBTL1. The analysis deals with novel ligands and targets, but applies widespread computational approaches and intuitive comparison metrics that makes this study compatible with and incremental to earlier large scale accounts on the efficiency of affinity predictors. Ultimately, this study has revealed three top performers, far ahead of the other techniques, including two scoring functions, PMF04 and PLP, along with a simulation-based method MM-PB/SA. We discuss why some methods may perform better than others on this target class, the limits of their application, as well as how the efficiency of the most CPU-demanding techniques could be optimized.
Project description:Lysine methylation facilitates protein-protein interactions through the activity of methyllysine (Kme) "reader" proteins. Functions of Kme readers have historically been studied in the context of histone interactions, where readers aid in chromatin-templated processes such as transcription, DNA replication and repair. However, there is growing evidence that Kme readers also function through interactions with non-histone proteins. To facilitate expanded study of Kme reader activities, we developed a high-throughput binding assay to reveal the sequence determinants of Kme-driven protein interactions. The assay queries a degenerate methylated lysine-oriented peptide library (Kme-OPL) to identify the key residues that modulate reader binding. The assay recapitulated methyl order and amino acid sequence preferences associated with histone Kme readers. The assay also revealed methylated sequences that bound Kme readers with higher affinity than histones. Proteome-wide scoring was applied to assay results to help prioritize future study of Kme reader interactions. The platform was also used to design sequences that directed specificity among closely related reader domains, an application which may have utility in the development of peptidomimetic inhibitors. Furthermore, we used the platform to identify binding determinants of site-specific histone Kme antibodies and surprisingly revealed that only a few amino acids drove epitope recognition. Collectively, these studies introduce and validate a rapid, unbiased, and high-throughput binding assay for Kme readers, and we envision its use as a resource for expanding the study of Kme-driven protein interactions.
Project description:Many non-histone proteins are lysine methylated and a novel function of this modification is to trigger the proteolysis of methylated proteins. Here, we report that the methylated lysine 142 of DNMT1, a major DNA methyltransferase that preserves epigenetic inheritance of DNA methylation patterns during DNA replication, is demethylated by LSD1. A novel methyl-binding protein, L3MBTL3, binds the K142-methylated DNMT1 and recruits a novel CRL4DCAF5 ubiquitin ligase to degrade DNMT1. Both LSD1 and PHF20L1 act primarily in S phase to prevent DNMT1 degradation by L3MBTL3-CRL4DCAF5. Mouse L3MBTL3/MBT-1 deletion causes accumulation of DNMT1 protein, increased genomic DNA methylation, and late embryonic lethality. DNMT1 contains a consensus methylation motif shared by many non-histone proteins including E2F1, a key transcription factor for S phase. We show that the methylation-dependent E2F1 degradation is also controlled by L3MBTL3-CRL4DCAF5. Our studies elucidate for the first time a novel mechanism by which the stability of many methylated non-histone proteins are regulated.
Project description:SOX2 is a dose-dependent master stem cell protein that controls the self-renewal and pluripotency or multipotency of embryonic stem (ES) cells and many adult stem cells. We have previously found that SOX2 protein is monomethylated at lysine residues 42 and 117 by SET7 methyltransferase to promote SOX2 proteolysis, whereas LSD1 and PHF20L1 act on both methylated Lys-42 and Lys-117 to prevent SOX2 proteolysis. However, the mechanism by which the methylated SOX2 protein is degraded remains unclear. Here, we report that L3MBTL3, a protein with the malignant-brain-tumor (MBT) methylation-binding domain, is required for SOX2 proteolysis. Our studies showed that L3MBTL3 preferentially binds to the methylated Lys-42 in SOX2, although mutation of Lys-117 also partially reduces the interaction between SOX2 and L3MBTL3. The direct binding of L3MBTL3 to the methylated SOX2 protein leads to the recruitment of the CRL4DCAF5 ubiquitin E3 ligase to target SOX2 protein for ubiquitin-dependent proteolysis. Whereas loss of either LSD1 or PHF20L1 destabilizes SOX2 protein and impairs the self-renewal and pluripotency of mouse ES cells, knockdown of L3MBTL3 or DCAF5 is sufficient to restore the protein levels of SOX2 and rescue the defects of mouse ES cells caused by LSD1 or PHF20L1 deficiency. We also found that retinoic acid-induced differentiation of mouse ES cells is accompanied by the enhanced degradation of the methylated SOX2 protein at both Lys-42 and Lys-117. Our studies provide novel insights into the mechanism by which the methylation-dependent degradation of SOX2 protein is controlled by the L3MBTL3-CRL4DCAF5 ubiquitin ligase complex.
Project description:The discovery of inhibitors of methyl- and acetyl-binding domains has provided evidence for the 'druggability' of epigenetic effector molecules. The small-molecule probe UNC1215 prevents methyl-dependent protein-protein interactions by engaging the aromatic cage of MBT domains and, with lower affinity, Tudor domains. Using a library of tagged UNC1215 analogs, we screened a protein-domain microarray of human methyllysine effector molecules to rapidly detect compounds with new binding profiles with either increased or decreased specificity. Using this approach, we identified a compound (EML405) that acquired a novel interaction with the Tudor-domain-containing protein Spindlin1 (SPIN1). Structural studies facilitated the rational synthesis of SPIN1 inhibitors with increased selectivity (EML631-633), which engage SPIN1 in cells, block its ability to 'read' H3K4me3 marks and inhibit its transcriptional-coactivator activity. Protein microarrays can thus be used as a platform to 'target-hop' and identify small molecules that bind and compete with domain-motif interactions.
Project description:Human L3MBTL1, which contains three malignant brain tumor (MBT) repeats, binds monomethylated and dimethylated lysines, but not trimethylated lysines, in several histone sequence contexts. In crystal structures of L3MBTL1 complexes, the monomethyl- and dimethyllysines insert into a narrow and deep cavity of aromatic residue-lined pocket 2, while a proline ring inserts into shallower pocket 1. We have also engineered a single Y to E substitution within the aromatic cage of the BPTF PHD finger, resulting in a reversal of binding preference from trimethyl- to dimethyllysine in an H3K4 sequence context. In both the "cavity insertion" (L3MBTL1) and "surface groove" (PHD finger) modes of methyllysine recognition, a carboxylate group both hydrogen bonds and ion pairs to the methylammonium proton. Our structural and binding studies of these two modules provide insights into the molecular principles governing the decoding of lysine methylation states, thereby highlighting a methylation state-specific layer of histone mark readout impacting on epigenetic regulation.
Project description:Notch signaling is an evolutionarily conserved signal transduction pathway that is essential for metazoan development. Upon ligand binding, the Notch intracellular domain (NOTCH ICD) translocates into the nucleus and forms a complex with the transcription factor RBPJ (also known as CBF1 or CSL) to activate expression of Notch target genes. In the absence of a Notch signal, RBPJ acts as a transcriptional repressor. Using a proteomic approach, we identified L3MBTL3 (also known as MBT1) as a novel RBPJ interactor. L3MBTL3 competes with NOTCH ICD for binding to RBPJ In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (also known as LSD1) to the enhancers of Notch target genes, leading to H3K4me2 demethylation and to transcriptional repression. Importantly, in vivo analyses of the homologs of RBPJ and L3MBTL3 in Drosophila melanogaster and Caenorhabditis elegans demonstrate that the functional link between RBPJ and L3MBTL3 is evolutionarily conserved, thus identifying L3MBTL3 as a universal modulator of Notch signaling in metazoans.