Journal: Genes & Diseases

Article Title: Role of post-translational modification of the Y box binding protein 1 in human cancers

doi: 10.1016/j.gendis.2015.05.001

Figure Lengend Snippet: Known and confirmed post-translational modifications in human YBX1.

Article Snippet: S136, T271 , CTD , Phosphorylation , – , Kettenbach et al 2011.

Techniques: Phospho-proteomics

Journal: Genes & Diseases

Article Title: Role of post-translational modification of the Y box binding protein 1 in human cancers

doi: 10.1016/j.gendis.2015.05.001

Figure Lengend Snippet: Predicted but unconfirmed post-translational modifications in human YBX1.

Article Snippet: S136, T271 , CTD , Phosphorylation , – , Kettenbach et al 2011.

Techniques: Phospho-proteomics

Journal: Nucleic Acids Research

Article Title: Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly

doi: 10.1093/nar/gkac451

Figure Lengend Snippet: Schematic representation. ( A ) Domain organization of Spt6 and its variants used in this study. Residues for the N’ terminus, core, tSH2 domain and different constructs of Spt6 are indicated. The core consists of five domains: HtH, helix-turn-helix domain; YqgF, looks similar to the RuvC catalytical domain; HhH, helix-hairpin-helix domain; DLD, death-like domain; S1 domain. Pictograms correspond to the flexible N’ terminus, core, and tSH2 domain. ( B ) Cartoon representation of histones and DNA used for nucleosome reconstitution, binding and assembly assay. ( C ) Peptide sequences of Pol II Rpb1 linker and CTD used in binding studies. The residues of the peptides in red indicate phosphorylations

Article Snippet: 5,6-FAM-labelled peptides corresponding to the N-terminal region of Spt6 ( S.c . Spt6 residues 257–289), phosphorylated Pol II Rpb1 linker (GGVTPpYSNESGLVNADLDVKDELMFpSPLVDSGS), and tyrosine-phosphorylated Pol II CTD (PSpYSPTSPSpYSPTSPS) were purchased from Caslo ApS.

Techniques: Construct, Binding Assay

Journal: Nucleic Acids Research

Article Title: Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly

doi: 10.1093/nar/gkac451

Figure Lengend Snippet: N-terminal region of Spt6 disrupts binding with Pol II and interacts with tSH2 domain and Spt6 core when added in trans. ( A ) Fluorescence anisotropy titration of phosphorylated Rpb1 CTD and Rpb1 linker peptides with Spt6 and ΔN-Spt6. Left panel – Rpb1 CTD binding with Spt6 lacking the flexible N-terminal region (cyan) K D ∼ 6.7 ± 1.56 μM; Spt6 (violet) K D ∼ n.d., not detected. Right panel – Rpb1 linker binding with Spt6 lacking the flexible N-terminal region (cyan) K D ∼ 11 ± 2.2 nM, Spt6 (violet) K D ∼ 290 ± 70 nM. Data represent means ± SD of technical triplicates. Normalized fluorescence anisotropy is plotted as a function of protein concentration. The data were normalized for visualization purposes and the experimental isotherms were fitted to a single-site saturation with non-specific binding model. ( B ) Fluorescence anisotropy titration shows that (left panel) N’ terminus binds to wild-type tSH2 (purple) K D ∼ 0.79 ± 0.20 μM, tSH2 variant with K1355A, K1435A double amino-acid substitutions (blue) K D ∼ 0.67 ± 0.39 μM, tSH2 variant with R1282H substitution (grey) K D ∼ n.d., not detected. ΔN-Spt6-ΔC associates with its N’ terminus (added in trans) with a K D ∼ 1.5 ± 0.7 μM (black, right panel). Points represent the mean ± SD of technical triplicates. The data were normalized for visualization purposes and the experimental isotherms were fitted to a single-site saturation with non-specific binding model

Article Snippet: 5,6-FAM-labelled peptides corresponding to the N-terminal region of Spt6 ( S.c . Spt6 residues 257–289), phosphorylated Pol II Rpb1 linker (GGVTPpYSNESGLVNADLDVKDELMFpSPLVDSGS), and tyrosine-phosphorylated Pol II CTD (PSpYSPTSPSpYSPTSPS) were purchased from Caslo ApS.

Techniques: Binding Assay, Fluorescence, Titration, Protein Concentration, Variant Assay

Journal: Nucleic Acids Research

Article Title: Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly

doi: 10.1093/nar/gkac451

Figure Lengend Snippet: Cryo-EM structure of Spt6 and comparison with other structures of Spt6. ( A ) Graphical representation of construct used for Cryo-EM studies. ( B ) Local resolution estimation, shading from red to blue indicates local resolution according to colour gradient. Domains are identified. ( C ) Resolution estimated using gold-standard FSC. Resolution is given for FSC 0.143. Flexibile regions (the N-terminal part of the central helix and associated regions) were masked out. ( D ) Electron density map variability of reconstituted ΔN-Spt6. Overlay of two cryo-EM density maps highlighting extreme positions of the central helix in violet and light green. The remaining parts of Spt6 are virtually identical and are shown in grey. For detailed comparison, see . ( E ) Overlay of unbound and Pol II-bound Spt6 structures. The cryo-EM structure of Spt6 bound to Pol II (violet, PDB ID: 6TED, ) and the crystal structure of the free form of Spt6 (light green, PDB ID: 3PSF, ). The largest conformational changes are observed for the highlighted central helix, which correspond to the variability of the cryo-EM density maps shown in (D).

Article Snippet: 5,6-FAM-labelled peptides corresponding to the N-terminal region of Spt6 ( S.c . Spt6 residues 257–289), phosphorylated Pol II Rpb1 linker (GGVTPpYSNESGLVNADLDVKDELMFpSPLVDSGS), and tyrosine-phosphorylated Pol II CTD (PSpYSPTSPSpYSPTSPS) were purchased from Caslo ApS.

Techniques: Cryo-EM Sample Prep, Comparison, Construct

Journal: Nucleic Acids Research

Article Title: Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly

doi: 10.1093/nar/gkac451

Figure Lengend Snippet: SAXS analysis of Spt6. Experimental SAXS profile of Spt6-ΔC ( A , top), Spt6 ( B , top), ΔN-Spt6 ( C , top) and theoretical scattering (red trace) calculated for model structures shown in bottom panels. ( D ) Structure of the Pol II–DSIF–PAF–SPT6 complex showing mutual orientation between the domains of SPT6 and Pol II. Pol II, pink; Rpb4, magenta; Rpb7, navy blue; Spt4, light green; Spt5, dark green; Spt6, grey; Spt6-tSH2, orange. Template and nontemplate DNA strands shown in blue and cyan, respectively; RNA, red. Color coding of Spt4, Spt5, DNA and RNA analogous as in for easier orientation

Article Snippet: 5,6-FAM-labelled peptides corresponding to the N-terminal region of Spt6 ( S.c . Spt6 residues 257–289), phosphorylated Pol II Rpb1 linker (GGVTPpYSNESGLVNADLDVKDELMFpSPLVDSGS), and tyrosine-phosphorylated Pol II CTD (PSpYSPTSPSpYSPTSPS) were purchased from Caslo ApS.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Fragment-based Differential Targeting of PPI Stabilizer Interfaces

doi: 10.1021/acs.jmedchem.9b01942

Figure Lengend Snippet: Identification of fragments binding to the interface of 14-3-3σ with p53. (A) Fragment AZ-001. (B) Crystal structure of AZ-001 (yellow sticks) in complex with 14-3-3σ (white surface) and p53pT387 (orange sticks), PDB 6S40. The final 2 F o – F c electron density maps are shown as blue mesh. (C) Detailed view of the binding pocket of AZ-001. The most prominent interaction is a salt-bridge (dotted black line) between the amidine of AZ-001 and carboxyl group of E14 of 14-3-3σ. (D) Fragment AZ-002. (E) Crystal structure of AZ-002 (yellow sticks) in complex with 14-3-3σ (white surface) and p53pT387 (orange sticks), PDB 6RWI. The final 2 F o – F c electron density maps are shown as blue mesh. (F) Detailed view of the binding pocket of AZ-002. Also with AZ-002, the most prominent interaction is a salt-bridge (dotted black line) between the amidine of AZ-002 and carboxyl group of E14 of 14-3-3σ.

Article Snippet: CTD Thr387 phosphorylated 32-mer peptide of p53 (AnaSpec) was immobilized onto a CMD200 M sensor chip (Xantec Bioanalytics) by using standard NHS/EDC amine-coupling chemistry.

Techniques: Binding Assay

Journal: Science Advances

Article Title: Structure and noncanonical Cdk8 activation mechanism within an Argonaute-containing Mediator kinase module

doi: 10.1126/sciadv.abd4484

Figure Lengend Snippet: ( A ) Structural organization of Med12. The first and second helices (orange ribbon) in Med12N are labeled as H1 and H2, respectively. Five HEAT domains (Med12HEAT) are shown in transparent surface. ( B ) Domain organization of Med12. The N- and C-terminal regions of Med12 (Med12N and Med12C) that form interactions with Cdk8/CycC and Med13, respectively, are indicated. Colors are as in (A). ( C ) Interactions of Med12 with Cdk8, CycC, and Med13. Cdk8, CycC, and Med13 are shown in colored surface representations. ( D ) The Med12 N-terminal region (residues 1 to 105) associates with Cdk8/CycC. GST-Med12 fragments in Escherichia coli lysates as indicated were immobilized on glutathione Sepharose beads and incubated with yeast cell lysate (CycC-TAP/Med12Δ/Med13Δ) containing Cdk8/CycC. ( E ) Kinase activity of yeast Cdk8/CycC stimulated by GST-Med12-(1–105). Phosphorylation of GST-CTD-6xHis was detected by the antibody that recognizes phosphorylated Ser 5 of CTD. For GST-Med12-(1–105), 250 ng (+) or 1 μg (++) of protein was used in the reactions. ( F ) Immunoprecipitation (IP) assay. Deletion of the C-terminal region (residues 1346 to 1427) of Med12 caused loss of Med13 from CKM.

Article Snippet: The antibody that recognizes phosphorylated Ser 5 of CTD (GenScript, A10634) was used to detect CTD phosphorylation.

Techniques: Labeling, Incubation, Activity Assay, Phospho-proteomics, Immunoprecipitation