Structural basis of SETD6-mediated regulation of the NF-kB network via methyl-lysine signaling

Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.
Nucleic Acids Research (Impact Factor: 9.11). 04/2011; 39(15):6380-9. DOI: 10.1093/nar/gkr256
Source: PubMed


SET domain containing 6 (SETD6) monomethylates the RelA subunit of nuclear factor kappa B (NF-κB). The ankyrin repeats of G9a-like protein (GLP) recognizes RelA monomethylated at Lys310. Adjacent to Lys310 is Ser311, a known phosphorylation site of RelA. Ser311 phosphorylation inhibits Lys310 methylation by SETD6 as well as binding of Lys310me1 by GLP. The structure of SETD6 in complex with RelA peptide containing the methylation site, in the presence of S-adenosyl-L-methionine, reveals a V-like protein structure and suggests a model for NF-κB binding to SETD6. In addition, structural modeling of the GLP ankyrin repeats bound to Lys310me1 peptide provides insight into the molecular basis for inhibition of Lys310me1 binding by Ser311 phosphorylation. Together, these findings provide a structural explanation for a key cellular signaling pathway centered on RelA Lys310 methylation, which is generated by SETD6 and recognized by GLP, and incorporate a methylation-phosphorylation switch of adjacent lysine and serine residues. Finally, SETD6 is structurally similar to the Rubisco large subunit methyltransferase. Given the restriction of Rubisco to plant species, this particular appearance of the protein lysine methyltransferase has been evolutionarily well conserved.

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    • "All ternary structures of SET domain methyltransferases in complex with cofactor and substrate have the substrate peptide lying in a narrow groove formed by the I-SET domain on one side, and the post-SET domain on the opposite side. This peptide binding groove is responsible for substrate sequence specificity and positions the substrate lysine side chain into a conserved channel that goes deep into the core of the SET domain and meets the cofactor at the conserved active site as described above (Figure 2) ([38,43,44,47,48,49,50,51,52] and PDB code 4AU7). The width of the peptide-binding groove is critical for catalysis, and the distance separating the two edges is between 7.2 and 7.8 Å across all available structures of active SET domain constructs (see materials and methods section for details). "
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    ABSTRACT: Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.
    PLoS ONE 12/2013; 8(12):e83737. DOI:10.1371/journal.pone.0083737 · 3.23 Impact Factor
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    • "WHSC1L1 Homo sapiens 2DAQ PWWP (NMR) SETD1A Homo sapiens 3S8S RRM SETD3 Homo sapiens 3SMT SAM 3QXY SAM N-lysine methyltransferase SETD6 Homo sapiens 3RC0 SAM N-lysine methyltransferase [378] "
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    Current pharmaceutical design 09/2012; 19(4). DOI:10.2174/138161213804581918 · 3.45 Impact Factor
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    • "We have recently reported that SETD6 mono-methylates RelA on lysine 310, leading to repression of RelA target genes [14,17]. Because RelA is the only SETD6 substrate known to date, we used the protein array to identify additional substrates of SETD6. "
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    ABSTRACT: ABSTRACT: Signaling via protein lysine methylation has been proposed to play a central role in the regulation of many physiologic and pathologic programs. In contrast to other post-translational modifications such as phosphorylation, proteome-wide approaches to investigate lysine methylation networks do not exist. In the current study, we used the ProtoArray® platform, containing over 9,500 human proteins, and developed and optimized a system for proteome-wide identification of novel methylation events catalyzed by the protein lysine methyltransferase (PKMT) SETD6. This enzyme had previously been shown to methylate the transcription factor RelA, but it was not known whether SETD6 had other substrates. By using two independent detection approaches, we identified novel candidate substrates for SETD6, and verified that all targets tested in vitro and in cells were genuine substrates. We describe a novel proteome-wide methodology for the identification of new PKMT substrates. This technological advance may lead to a better understanding of the enzymatic activity and substrate specificity of the large number (more than 50) PKMTs present in the human proteome, most of which are uncharacterized.
    Epigenetics & Chromatin 10/2011; 4(1):19. DOI:10.1186/1756-8935-4-19 · 5.33 Impact Factor
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