Structural and biochemical studies of human lysine methyltransferase Smyd3 reveal the important functional roles of its post-SET and TPR domains and the regulation of its activity by DNA binding.
ABSTRACT: The SET- and MYND-domain containing (Smyd) proteins constitute a special subfamily of the SET-containing lysine methyltransferases. Here we present the structure of full-length human Smyd3 in complex with S-adenosyl-L-homocysteine at 2.8?Å resolution. Smyd3 affords the first example that other region(s) besides the SET domain and its flanking regions participate in the formation of the active site. Structural analysis shows that the previously uncharacterized C-terminal domain of Smyd3 contains a tetratrico-peptide repeat (TPR) domain which together with the SET and post-SET domains forms a deep, narrow substrate binding pocket. Our data demonstrate the important roles of both TPR and post-SET domains in the histone lysine methyltransferase (HKMT) activity of Smyd3, and show that the hydroxyl group of Tyr239 is critical for the enzymatic activity. The characteristic MYND domain is located nearby to the substrate binding pocket and exhibits a largely positively charged surface. Further biochemical assays show that DNA binding of Smyd3 can stimulate its HKMT activity and the process may be mediated via the MYND domain through direct DNA binding.
Project description:SMYD3, a SET and MYND domain containing lysine methyltransferase, catalyzes the transfer of the methyl group from a methyl donor onto the N? group of a lysine residue in the substrate protein. Methylation of MAP3 kinase kinase (MAP3K2) by SMYD3 has been implicated in Ras-driven tumorigenesis. The crystal structure of SMYD3 in complex with MAP3K2 peptide reveals a shallow hydrophobic pocket (P-2), which accommodates the binding of a phenylalanine residue at the -2 position of the substrate (F258) is a crucial determinant of substrate specificity of SMYD3. To better understand the substrate preference of SMYD3 at the -2 position, molecular dynamics (MD) simulations and the MM/GBSA method were performed on the crystal structure of SMYD3-MAP3K2 complex (PDB: 5EX0) after substitution of F258 residue of MAP3K2 to each of the other 19 natural residues, respectively. Binding free energy calculations reveal that the P-2 pocket prefers an aromatic hydrophobic group and none of the substitutions behave better than the wild-type phenylalanine residue does. Furthermore, we investigated the structure-activity relationships (SAR) of a series of non-natural phenylalanine derivative substitutions at the -2 position and found that quite a few modifications on the sidechain of F258 residue could strengthen its binding to the P-2 pocket of SMYD3. These explorations provide insights into developing novel SMYD3 inhibitors with high potency and high selectivity against MAP3K2 and cancer.
Project description:Hepatitis C virus (HCV) is a considerable global health and economic burden. The HCV nonstructural protein (NS) 5A is essential for the viral life cycle. The ability of NS5A to interact with different host and viral proteins allow it to manipulate cellular pathways and regulate viral processes, including RNA replication and virus particle assembly. As part of a proteomic screen, we identified several NS5A-binding proteins, including the lysine methyltransferase SET and MYND domain containing protein 3 (SMYD3). We confirmed the interaction in the context of viral replication by co-immunoprecipitation and co-localization studies. Mutational analyses revealed that the MYND-domain of SMYD3 and domain III of NS5A are required for the interaction. Overexpression of SMYD3 resulted in decreased intracellular and extracellular virus titers, whilst viral RNA replication remained unchanged, suggesting that SMYD3 negatively affects HCV particle production in a NS5A-dependent manner.
Project description:The SET and MYND Domain (SMYD) proteins comprise a unique family of multi-domain SET histone methyltransferases that are implicated in human cancer progression. Here we report an analysis of the crystal structure of the full length human SMYD3 in a complex with an analog of the S-adenosyl methionine (SAM) methyl donor cofactor. The structure revealed an overall compact architecture in which the "split-SET" domain adopts a canonical SET domain fold and closely assembles with a Zn-binding MYND domain and a C-terminal superhelical 9 ?-helical bundle similar to that observed for the mouse SMYD1 structure. Together, these structurally interlocked domains impose a highly confined binding pocket for histone substrates, suggesting a regulated mechanism for its enzymatic activity. Our mutational and biochemical analyses confirm regulatory roles of the unique structural elements both inside and outside the core SET domain and establish a previously undetected preference for trimethylation of H4K20.
Project description:Macromolecular complexes are essential to intracellular signal transduction by creating signaling niches and enabling a chain of reactions that transmit external signals into various cellular responses. Analysis of SMYD3 interactome indicates this protein lysine methyltransferase might be involved in calcium dependent signaling pathways through forming complexes with the phospholipase PLCB3, calcium/calmodulin dependent kinase CAMK2B, or calcineurin inhibitor RCAN3. SMYD3 is well-known as a histone H3K4 methyltransferase involved in epigenetic transcriptional regulation; however, any roles SMYD3 may play in signaling transduction remain unknown. KEGG pathway enrichment analysis reveals the SMYD3 interacting proteins are overrepresented in several signaling pathways such as estrogen signaling pathway, NOD-like receptor signaling pathway, and WNT signaling pathway. Sequence motif scanning reveals a significant enrichment of PXLXP motif in SMYD3 interacting proteins. The MYND domain of SMYD3 is known to bind to the PXLXP motif. The enrichment of the PXLXP motif suggests that the MYND domain is likely to be a key interaction module that mediates formation of some SMYD3 complexes. The presence of the PXLXP motifs in PLCB3 and CAMK2B indicates the potential role of the MYND domain in mediating complex formation in signaling. The structural basis of SMYD3 MYND domain-mediated interactions is unknown. The only available MYND-peptide complex structure suggests the MYND domain-mediated interaction is likely transient and dynamic. The transient nature will make this domain well-suited to mediate signaling transduction processes where it may allow rapid responses to cellular perturbations and changes in environment.
Project description:Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.
Project description:SMYD3 is a SET domain-containing N-lysine methyltransferase associated with multiple cancers. Its reported substrates include histones (H3K4 and H4K5), vascular endothelial growth factor receptor 1 (VEGFR1 Lys(831)) and MAP3 kinase kinase (MAP3K2 Lys(260)). To reveal the structural basis for substrate preference and the catalytic mechanism of SMYD3, we have solved its co-crystal structures with VEGFR1 and MAP3K2 peptides. Our structural and biochemical analyses show that MAP3K2 serves as a robust substrate of SMYD3 because of the presence of a phenylalanine residue at the -2 position. A shallow hydrophobic pocket on SMYD3 accommodates the binding of the phenylalanine and promotes efficient catalytic activities of SMYD3. By contrast, SMYD3 displayed a weak activity toward a VEGFR1 peptide, and the location of the acceptor lysine in the folded kinase domain of VEGFR1 requires drastic conformational rearrangements for juxtaposition of the acceptor lysine with the enzymatic active site. Our results clearly revealed structural determinants for the substrate preference of SMYD3 and provided mechanistic insights into lysine methylation of MAP3K2. The knowledge should be useful for the development of SMYD3 inhibitors in the fight against MAP3K2 and Ras-driven cancer.
Project description:Mammalian early embryonic development is controlled by a unique program of gene expression, and involves epigenetic reprogramming of histone modifications and DNA methylation. SET and MYND domain-containing protein 3 (SMYD3) is a histone H3 lysine 4 methyltransferase that plays important roles in transcription regulation. The expression of SMYD3 has been studied in some cancer cell lines. However, its expression in oocytes and embryos has not previously been reported. Here, we detected the SMYD3 mRNA and found that it was expressed throughout bovine oocyte in vitro maturation and early embryonic development. Microinjection of SMYD3 siRNA at germinal vesicle stage decreased the transcription level of NANOG, and blocked the development of in vitro fertilization embryos at 4-8 cell stage. Conversely, Microinjection of SMYD3 siRNA at pronuclear stage did not affect early embryonic development. Our findings suggest that SMYD3 regulates the expression of NANOG, and plays an essential role in bovine early embryonic development.
Project description:SMYD3 is a SET-domain-containing methyltransferase that catalyzes the transfer of methyl groups onto lysine residues of substrate proteins. Methylation of MAP3K2 by SMYD3 has been implicated in Ras-driven tumorigenesis, which makes SMYD3 a potential target for cancer therapy. Of all SMYD family proteins, SMYD3 adopt a closed conformation in a crystal structure. Several studies have suggested that the conformational changes between the open and closed forms may regulate the catalytic activity of SMYD3. In this work, we carried out extensive molecular dynamics simulations on a series of complexes with a total of 21 μs sampling to investigate the conformational changes of SMYD3 and unveil the molecular mechanisms. Based on the C-terminal domain movements, the simulated models could be depicted in three different conformational states: the closed, intermediate and open states. Only in the case that both the methyl donor binding pocket and the target lysine-binding channel had bound species did the simulations show SMYD3 maintaining its conformation in the closed state, indicative of a synergetic effect of the cofactors and target lysine on regulating the conformational change of SMYD3. In addition, we performed analyses in terms of structure and energy to shed light on how the two regions might regulate the C-terminal domain movement. This mechanistic study provided insights into the relationship between the conformational change and the methyltransferase activity of SMYD3. The more complete understanding of the conformational dynamics developed here together with further work may lay a foundation for the rational drug design of SMYD3 inhibitors.
Project description:We report herein the discovery of isoxazole amides as potent and selective SET and MYND Domain-Containing Protein 3 (SMYD3) inhibitors. Elucidation of the structure-activity relationship of the high-throughput screening (HTS) lead compound <b>1</b> provided potent and selective SMYD3 inhibitors. The SAR optimization, cocrystal structures of small molecules with SMYD3, and mode of inhibition (MOI) characterization of compounds are described. The synthesis and biological and pharmacokinetic profiles of compounds are also presented.
Project description:SET and MYND domain containing protein 3 (SMYD3) is a histone methyltransferase, which has been implicated in cell growth and cancer pathogenesis. Increasing evidence suggests that SMYD3 can influence distinct oncogenic processes by acting as a gene-specific transcriptional regulator. However, the mechanistic aspects of SMYD3 transactivation and whether SMYD3 acts in concert with other transcription modulators remain unclear. Here, we show that SMYD3 interacts with the human positive coactivator 4 (PC4) and that such interaction potentiates a group of genes whose expression is linked to cell proliferation and invasion. SMYD3 cooperates functionally with PC4, because PC4 depletion results in the loss of SMYD3-mediated H3K4me3 and target gene expression. Individual depletion of SMYD3 and PC4 diminishes the recruitment of both SMYD3 and PC4, indicating that SMYD3 and PC4 localize at target genes in a mutually dependent manner. Artificial tethering of a SMYD3 mutant incapable of binding to its cognate elements and interacting with PC4 to target genes is sufficient for achieving an active transcriptional state in SMYD3-deficient cells. These observations suggest that PC4 contributes to SMYD3-mediated transactivation primarily by stabilizing SMYD3 occupancy at target genes. Together, these studies define expanded roles for SMYD3 and PC4 in gene regulation and provide an unprecedented documentation of their cooperative functions in stimulating oncogenic transcription.