SH3 domains are protein modules that mediate protein-protein interactions in many eukaryotic signal transduction pathways. The majority of SH3 domains studied thus far act by binding to proline-rich ...sequences in partner proteins, but a growing number of studies have revealed alternative recognition mechanisms. We have comprehensively surveyed the specificity landscape of human SH3 domains in an unbiased manner using peptide-phage display and deep sequencing. Based on ∼70,000 unique binding peptides, we obtained 154 specificity profiles for 115 SH3 domains, which reveal that roughly half of the SH3 domains exhibit non-canonical specificities and collectively recognize a wide variety of peptide motifs, most of which were previously unknown. Crystal structures of SH3 domains with two distinct non-canonical specificities revealed novel peptide-binding modes through an extended surface outside of the canonical proline-binding site. Our results constitute a significant contribution toward a complete understanding of the mechanisms underlying SH3-mediated cellular responses.
•We obtained a map of 154 specificity profiles for 115 human SH3 domains•Half of the SH3 domains exhibit a variety of non-canonical specificities•Crystal structures reveal novel peptide-binding modes for SH3 domains•The specificity map can guide further structural and biological studies
The 154 specificity profiles obtained for 115 SH3 domains reveal that roughly half of the domains exhibit non-canonical specificities. The wide variety of atypical specificities suggests diverse strategies for recognizing peptide ligands, and crystal structures show the details for two novel SH3-peptide-binding modes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
CFP1 is a CXXC domain-containing protein and an essential component of the SETD1 histone H3K4 methyltransferase complex. CXXC domain proteins direct different chromatin-modifying activities to ...various chromatin regions. Here, we report crystal structures of the CFP1 CXXC domain in complex with six different CpG DNA sequences. The crescent-shaped CFP1 CXXC domain is wedged into the major groove of the CpG DNA, distorting the B-form DNA, and interacts extensively with the major groove of the DNA. The structures elucidate the molecular mechanism of the non-methylated CpG-binding specificity of the CFP1 CXXC domain. The CpG motif is confined by a tripeptide located in a rigid loop, which only allows the accommodation of the non-methylated CpG dinucleotide. Furthermore, we demonstrate that CFP1 has a preference for a guanosine nucleotide following the CpG motif.
Structure and Biochemical Functions of SIRT6 Pan, Patricia W.; Feldman, Jessica L.; Devries, Mark K. ...
Journal of biological chemistry/The Journal of biological chemistry,
04/2011, Volume:
286, Issue:
16
Journal Article
Peer reviewed
Open access
SIRT6 is a member of the evolutionarily conserved sirtuin family of NAD+-dependent protein deacetylases and functions in genomic stability and transcriptional control of glucose metabolism. Early ...reports suggested that SIRT6 performs ADP-ribosylation, whereas more recent studies have suggested that SIRT6 functions mainly as a histone deacetylase. Thus, the molecular functions of SIRT6 remain uncertain. Here, we perform biochemical, kinetic, and structural studies to provide new mechanistic insight into the functions of SIRT6. Utilizing three different assays, we provide biochemical and kinetic evidence that SIRT6-dependent histone deacetylation produces O-acetyl-ADP-ribose but at a rate ∼1,000 times slower than other highly active sirtuins. To understand the molecular basis for such low deacetylase activity, we solved the first crystal structures of this class IV sirtuin in complex with ADP-ribose and the non-hydrolyzable analog of O-acetyl-ADP-ribose, 2′-N-acetyl-ADP-ribose. The structures revealed unique features of human SIRT6, including a splayed zinc-binding domain and the absence of a helix bundle that in other sirtuin structures connects the zinc-binding motif and Rossmann fold domain. SIRT6 also lacks the conserved, highly flexible, NAD+-binding loop and instead contains a stable single helix. These differences led us to hypothesize that SIRT6, unlike all other studied sirtuins, would be able to bind NAD+ in the absence of an acetylated substrate. Indeed, we found that SIRT6 binds NAD+ with relatively high affinity (Kd = 27 ± 1 μm) in the absence of an acetylated substrate. Isothermal titration calorimetry and tryptophan fluorescence binding assays suggested that ADP-ribose and NAD+ induce different structural perturbations and that NADH does not bind to SIRT6. Collectively, these new insights imply a unique activating mechanism and/or the possibility that SIRT6 could act as an NAD+ metabolite sensor.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
MLL3 and MLL4 are two closely related members of the SET1/MLL family of histone H3K4 methyltransferases and are responsible for monomethylating histone H3K4 on enhancers, which are essential in ...regulating cell-type-specific gene expression. Mutations of MLL3 or MLL4 have been reported in different types of cancer. Recently, the PHD domains of MLL3/4 have been reported to recruit the MLL3/4 complexes to their target genes by binding to histone H4 during the NT2/D1 stem cell differentiation. Here we show that an extended PHD domain (ePHD
) involving the sixth PHD domain and its preceding zinc finger in MLL3 and MLL4 specifically recognizes an H4H18-containing histone H4 fragment and that modifications of residues surrounding H4H18 modulate H4 binding to MLL3/4. Our in vitro methyltransferase assays and cellular experiments further reveal that the interaction between ePHD
of MLL3/4 and histone H4 is required for their nucleosomal methylation activity and MLL4-mediated neuronal differentiation of NT2/D1 cells.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
G9a-like protein (GLP) and G9a are highly homologous protein lysine methyltransferases (PKMTs) sharing approximately 80% sequence identity in their catalytic domains. GLP and G9a form a heterodimer ...complex and catalyze mono- and dimethylation of histone H3 lysine 9 and nonhistone substrates. Although they are closely related, GLP and G9a possess distinct physiological and pathophysiological functions. Thus, GLP or G9a selective small-molecule inhibitors are useful tools to dissect their distinct biological functions. We previously reported potent and selective G9a/GLP dual inhibitors including UNC0638 and UNC0642. Here we report the discovery of potent and selective GLP inhibitors including 4 (MS0124) and 18 (MS012), which are >30-fold and 140-fold selective for GLP over G9a and other methyltransferases, respectively. The cocrystal structures of GLP and G9a in the complex with either 4 or 18 displayed virtually identical binding modes and interactions, highlighting the challenges in structure-based design of selective inhibitors for either enzyme.
Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone ...targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the ...discovery of (R)-PFI-2—a first-in-class, potent (K ᵢᵃᵖᵖ = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7—and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S -adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as a ...candidate oncogene owing to its overexpression in several types of cancer. Selective PRMT4 inhibitors are useful tools for clarifying the molecular events regulated by PRMT4 and for validating PRMT4 as a therapeutic target. Here, we report the discovery of TP-064, a potent, selective, and cell-active chemical probe of human PRMT4 and its co-crystal structure with PRMT4. TP-064 inhibited the methyltransferase activity of PRMT4 with high potency (half-maximal inhibitory concentration, IC
< 10 nM) and selectivity over other PRMT family proteins, and reduced arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155; IC
= 340 ± 30 nM) and Mediator complex subunit 12 (MED12; IC
= 43 ± 10 nM). TP-064 treatment inhibited the proliferation of a subset of multiple myeloma cell lines, with affected cells arrested in G1 phase of the cell cycle. TP-064 and its negative control (TP-064N) will be valuable tools to further investigate the biology of PRMT4 and the therapeutic potential of PRMT4 inhibition.
TXNIP (thioredoxin-interacting protein) negatively regulates the antioxidative activity of thioredoxin and participates in pleiotropic cellular processes. Its deregulation is linked to various human ...diseases, including diabetes, acute myeloid leukaemia and cardiovascular diseases. The E3 ubiquitin ligase Itch (Itchy homologue) polyubiquitinates TXNIP to promote its degradation via the ubiquitin-proteasome pathway, and this Itch-mediated polyubiquitination of TXNIP is dependent on the interaction of the four WW domains of Itch with the two PPxY motifs of TXNIP. However, the molecular mechanism of this interaction of TXNIP with Itch remains elusive. In the present study, we found that each of the four WW domains of Itch exhibited different binding affinities for TXNIP, whereas multivalent engagement between the four WW domains of Itch and the two PPxY motifs of TXNIP resulted in their strong binding avidity. Our structural analyses demonstrated that the third and fourth WW domains of Itch were able to recognize both PPxY motifs of TXNIP simultaneously, supporting a multivalent binding mode between Itch and TXNIP. Interestingly, the phosphorylation status on the tyrosine residue of the PPxY motifs of TXNIP serves as a molecular switch in its choice of binding partners and thereby downstream biological signalling outcomes. Phosphorylation of this tyrosine residue of TXNIP diminished the binding capability of PPxY motifs of TXNIP to Itch, whereas this phosphorylation is a prerequisite to the binding activity of TXNIP to SHP2 SH2 (Src homology 2) domain-containing protein tyrosine phosphatase 2 and their roles in stabilizing the phosphorylation and activation of CSK (c-Src tyrosine kinase).