PTENα/β, two variants of PTEN, play a key role in promoting tumor growth by interacting with WDR5 through their N-terminal extensions (NTEs). This interaction facilitates the recruitment of the SET1/MLL methyltransferase complex, resulting in histone H3K4 trimethylation and upregulation of oncogenes such as NOTCH3, which in turn promotes tumor growth. However, the molecular mechanism underlying this interaction has remained elusive. In this study, we determined the first crystal structure of PTENα-NTE in complex with WDR5, which reveals that PTENα utilizes a unique binding motif of a sequence SSSRRSS found in the NTE domain of PTENα/β to specifically bind to the WIN site of WDR5. Disruption of this interaction significantly impedes cell proliferation and tumor growth, highlighting the potential of the WIN site inhibitors of WDR5 as a way of therapeutic intervention of the PTENα/β associated cancers. These findings not only shed light on the important role of the PTENα/β-WDR5 interaction in carcinogenesis, but also present a promising avenue for developing cancer treatments that target this pathway.
The Type VI-D CRISPR-Cas system employs an RNA-guided RNase Cas13d with minimal targeting constraints to combat viral infections. This CRISPR system contains RspWYL1 as a unique accessory protein that plays a key role in boosting its effector function on target RNAs, but the mechanism behind this RspWYL1-mediated stimulation remains completely unexplored. Through structural and biophysical approaches, we reveal that the full-length RspWYL1 possesses a novel three-domain architecture and preferentially binds ssRNA with high affinity. Specifically, the N-terminus of RspWYL1 harbors a ribbon-helix-helix motif reminiscent of transcriptional regulators; the central WYL domain of RspWYL1 displays a Sm-like β-barrel fold; and the C-terminal domain of RspWYL1 primarily contributes to the dimerization of RspWYL1 and may regulate the RspWYL1 function via a large conformational change. Collectively, this study provides a first glimpse into the complex mechanism behind the RspWYL1-dictated boosting of target ssRNA cleavage in the Type VI-D CRISPR-Cas system.
SETD3 is a member of SET (Su(var)3-9, Enhancer of zeste, and Trithorax) domain protein superfamily and plays important roles in hypoxic pulmonary hypertension, muscle differentiation, and carcinogenesis. Recently, we have identified SETD3 as the actin-specific methyltransferase that methylates the N3 of His73 on β-actin. Here we present two structures of S-adenosyl-L-homocysteine-bound SETD3 in complex with either an unmodified β-actin peptide or its His-methylated variant. Structural analyses supported by the site-directed mutagenesis experiments and the enzyme activity assays indicated that the recognition and methylation of β-actin by SETD3 is highly sequence specific, and both SETD3 and β-actin adopt pronounce conformational changes upon binding to each other. In conclusion, the data show for the first time a catalytic mechanism of SETD3-mediated histidine methylation in β-actin, which not only throws light on protein histidine methylation phenomenon, but also facilitates the design of small molecule inhibitors of SETD3.
PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes.
Experiment: Expression test and purification of SETDB1 catalytic domain constructs’ for structural studies by X-ray crystallography. Aim: In the present section of this study, we focused on the development of efficient bacterial expression systems to produce large amounts of soluble SETDB1 catalytic domain for structural studies. This report involves a summary of expression test results of different fragments of SETDB1 and purification of various fusion proteins.
Abstract A large number of structurally diverse epigenetic reader proteins specifically recognize methylated lysine residues on histone proteins. Here we describe comparative thermodynamic, structural and computational studies on recognition of the positively charged natural trimethyllysine and its neutral analogues by reader proteins. This work provides experimental and theoretical evidence that reader proteins predominantly recognize trimethyllysine via a combination of favourable cation– π interactions and the release of the high-energy water molecules that occupy the aromatic cage of reader proteins on the association with the trimethyllysine side chain. These results have implications in rational drug design by specifically targeting the aromatic cage of readers of trimethyllysine.
The effects of chemical substitution on the crystal structure and superconducting properties of (${\mathrm{Pb}}_{0.5}$${\mathrm{Cd}}_{0.5}$)(Sr,Y,Ca${)}_{3}$${\mathrm{Cu}}_{2}$${\mathrm{O}}_{7\mathrm{\ensuremath{-}}\mathrm{\ensuremath{\delta}}}$ have been studied in (${\mathrm{Pb}}_{0.5}$${\mathrm{Cd}}_{0.5}$)${\mathrm{Sr}}_{2}$(${\mathrm{Y}}_{1\mathrm{\ensuremath{-}}\mathit{x}}$${\mathrm{Ca}}_{\mathit{x}}$)${\mathrm{Cu}}_{2}$${\mathrm{O}}_{7\mathrm{\ensuremath{-}}\mathrm{\ensuremath{\delta}}}$, (${\mathrm{Pb}}_{0.5}$${\mathrm{Cd}}_{0.5}$) ${\mathrm{Sr}}_{2\mathrm{\ensuremath{-}}\mathit{x}}$${\mathrm{Y}}_{1\mathrm{\ensuremath{-}}\mathit{u}+\mathit{x}}$${\mathrm{Ca}}_{\mathit{u}}$${\mathrm{Cu}}_{2}$${\mathrm{O}}_{7\mathrm{\ensuremath{-}}\mathrm{\ensuremath{\delta}}}$ (u=0.0,0.3,0.5), and (${\mathrm{Pb}}_{0.5}$${\mathrm{Cd}}_{0.5}$) ${\mathrm{Sr}}_{2\mathrm{\ensuremath{-}}\mathit{x}}$${\mathrm{Ca}}_{\mathit{v}+\mathit{x}}$${\mathrm{Y}}_{1\mathrm{\ensuremath{-}}\mathit{v}}$${\mathrm{Cu}}_{2}$${\mathrm{O}}_{7\mathrm{\ensuremath{-}}\mathrm{\ensuremath{\delta}}}$ (v=0.0,0.1,0.3). The single-phase region of (${\mathrm{Pb}}_{0.5}$${\mathrm{Cd}}_{0.5}$)${\mathrm{Sr}}_{3\mathrm{\ensuremath{-}}\mathit{x}\mathrm{\ensuremath{-}}\mathit{y}}$${\mathrm{Ca}}_{\mathit{x}}$${\mathrm{Y}}_{\mathit{y}}$${\mathrm{Cu}}_{2}$${\mathrm{O}}_{7\mathrm{\ensuremath{-}}\mathrm{\ensuremath{\delta}}}$ is 0\ensuremath{\le}x\ensuremath{\le}0.5 and 0.8\ensuremath{\le}y\ensuremath{\le}1.0. In (${\mathrm{Pb}}_{0.5}$${\mathrm{Cd}}_{0.5}$)${\mathrm{Sr}}_{2}$(${\mathrm{Y}}_{1\mathrm{\ensuremath{-}}\mathit{x}}$${\mathrm{Ca}}_{\mathit{x}}$)${\mathrm{Cu}}_{2}$${\mathrm{O}}_{7\mathrm{\ensuremath{-}}\mathrm{\ensuremath{\delta}}}$, the lattice parameter c anomalously decreases while a remains nearly unchanged with the substitution of Ca for Y (0\ensuremath{\le}x\ensuremath{\le}0.2).This phenomenon is related to the decrease of the oxygen content. The investigations of (${\mathrm{Pb}}_{0.5}$${\mathrm{Cd}}_{0.5}$) ${\mathrm{Sr}}_{2\mathrm{\ensuremath{-}}\mathit{x}}$${\mathrm{Y}}_{1\mathrm{\ensuremath{-}}\mathit{u}+\mathit{x}}$${\mathrm{Ca}}_{\mathit{u}}$${\mathrm{Cu}}_{2}$${\mathrm{O}}_{7\mathrm{\ensuremath{-}}\mathrm{\ensuremath{\delta}}}$ (u=0.0,0.3,0.5) reveal that it is difficult for Y to enter the Si site (2h) and Ca can enter the Sr site (2h). The chemical formula of (${\mathrm{Pb}}_{0.5}$${\mathrm{Cd}}_{0.5}$) ${\mathrm{Sr}}_{2\mathrm{\ensuremath{-}}\mathit{x}}$${\mathrm{Y}}_{1\mathrm{\ensuremath{-}}\mathit{u}+\mathit{x}}$${\mathrm{Ca}}_{\mathit{u}}$${\mathrm{Cu}}_{2}$${\mathrm{O}}_{7\mathrm{\ensuremath{-}}\mathrm{\ensuremath{\delta}}}$ (u=0.3,0.5) for the x>0 sides can be written as (${\mathrm{Pb}}_{0.5}$${\mathrm{Cd}}_{0.5}$)(${\mathrm{Sr}}_{2\mathrm{\ensuremath{-}}\mathit{x}}$${\mathrm{Ca}}_{\mathit{x}}$) (${\mathrm{Y}}_{1\mathrm{\ensuremath{-}}\mathit{u}+\mathit{x}}$${\mathrm{Ca}}_{\mathit{u}\mathrm{\ensuremath{-}}\mathit{x}}$) ${\mathrm{Cu}}_{2}$${\mathrm{O}}_{7\mathrm{\ensuremath{-}}\mathrm{\ensuremath{\delta}}}$ (u=0.3,0.5). Besides, we have also studied the relationships between the lattice parameters and chemical substitution, and found that the substitution of Ca for Sr in the Y site (1d) and Ca for Sr in the Sr site (2h) has different influence on the lattice parameters.The variations of lattice parameters with composition are linear for x0 or x>0. The \ensuremath{\Delta}c/\ensuremath{\Delta}x values are -0.12 \AA{} and -0.32 \AA{} for x\ensuremath{\le}0 and x\ensuremath{\ge}0, respectively.
Adenylation domain (A domain) is a model of non-ribosomal peptide synthetases (NRPs), responsible for binding and activating the substrates-amino acids.In this study, a pGEX-2T-sare0718 recombinant plasmid (the gene sare0718 was cloned from Salinispora arenicola CNS-205, S. arenicola CNS-205) was transformed and expressed as a protein GST-Sare0718.Fluorescence quenching (FQ) was conducted to investigate the binding of 20 common amino acids to GST-Sare0718, then isothermal titration calorimetry (ITC) also be used.The results of FQ revealed that intrinsic fluorescence of GST-Sare0718 is quenched steadily by addition of aspartic acid (Asp) and glutamine (Gln) through static quenching mechanism.The binding constants K a are Asp (1.504 ◊ 10 6 L/mol) = Gln (1.468 ◊ 10 5 L/mol).And there is one binding site on the protein GST-Sare0718.We confirmed Asp preference of GST-Sare0718 by ITC.Therefore, Asp is the specific substrate.In addition, the experimental data indicates that the prediction system, ìthe specificity-conferring codeî, is not suitable for marine actinomycetes.So it is urgent to set up a special predictive system for marine actinomycetes.
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