ncor id1 peptide  (LifeTein Inc)

Journal: Science Advances

Article Title: Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

doi: 10.1126/sciadv.abc6479

Figure Lengend Snippet: ( A ) REV-ERBβ and NCoR domain architecture. REV-ERBβ domains include DNA binding domain (DBD) and LBD. NCoR domains include repression domains (RDs) and the RID encompassing two ID motifs (ID1 and ID2). ( B ) Representative ITC thermograms and fitted curves of REV-ERBβ LBD titrated with NCoR ID1 or ID2 peptide in the presence or absence of heme with calculated binding affinities inset in each plot (error bars, uncertainty in each injection calculated by the NITPIC algorithm); each panel is representative of two or more independent experiments. ( C ) Representative peaks from 2D [ 1 H, 15 N]-TROSY-HSQC spectra of 15 N-labeled REV-ERBβ LBD with (orange/red peaks) or without heme (blue peaks) titrated with NCoR ID1 or ID2 peptide. ( D ) Residues with the largest chemical shift perturbation (CSP) changes or line broadening in the 2D [ 1 H, 15 N]-TROSY-HSQC spectra with addition of 2× ID1 or ID2 peptide [relative to the dimethyl sulfoxide (DMSO) vehicle spectrum] were mapped onto the crystal structure of heme-bound REV-ERBβ LBD [Protein Data Bank (PDB) 3CQV]. ppm, parts per million. DP, differential power.

Article Snippet: NCoR ID1 peptide (RTHRLITLADHICQIITQDFARN) and NCoR ID2 peptide (DPASNLGLEDIIRKALMGSFDDK) were purchased with >95% from LifeTein with N-terminal amidation and C-terminal acetylation for stability and prepared as 50 mM stocks in DMSO stored at −80°C.

Techniques: Binding Assay, Injection, Labeling

Journal: Science Advances

Article Title: Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

doi: 10.1126/sciadv.abc6479

Figure Lengend Snippet: Thermodynamic parameters of NCoR ID peptide binding to REV-ERBβ LBD. K D , binding affinity; Δ G , free energy of binding; Δ H , enthalpy of binding; temperature ( T ), constant at 25°C; and entropy (Δ S ) of binding.

Article Snippet: NCoR ID1 peptide (RTHRLITLADHICQIITQDFARN) and NCoR ID2 peptide (DPASNLGLEDIIRKALMGSFDDK) were purchased with >95% from LifeTein with N-terminal amidation and C-terminal acetylation for stability and prepared as 50 mM stocks in DMSO stored at −80°C.

Techniques: Binding Assay

Journal: Science Advances

Article Title: Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

doi: 10.1126/sciadv.abc6479

Figure Lengend Snippet: ( A ) Structure of REV-ERBβ LBD (light orange cartoon with the AF-2 surface in gray) cobound to heme (red sticks) and NCoR ID1 peptide (light purple cartoon) (PDB 6WMQ). ( B ) Structure of REV-ERBβ LBD (light orange cartoon with the AF-2 surface in gray) cobound to heme (red sticks) and NCoR ID2 peptide (dark blue cartoon) (PDB 6WMS). Insets to the left of the structures highlight the ID peptide CoRNR box motif residues (in bold) and conserved charge clamp interaction with K421. Omit maps (2 F o − F c , contoured at 1σ) for the ID peptides and heme are shown to the right of the structures with CoRNR box motif residues indicated.

Article Snippet: NCoR ID1 peptide (RTHRLITLADHICQIITQDFARN) and NCoR ID2 peptide (DPASNLGLEDIIRKALMGSFDDK) were purchased with >95% from LifeTein with N-terminal amidation and C-terminal acetylation for stability and prepared as 50 mM stocks in DMSO stored at −80°C.

Techniques:

Journal: Science Advances

Article Title: Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

doi: 10.1126/sciadv.abc6479

Figure Lengend Snippet: Representative ITC thermograms and fitted curves of apo ( A ) or heme-bound ( B ) REV-ERBβ LBD and NCoR RID constructs; data are representative of at least two experiments per condition (error bars, uncertainty in each injection calculated by the NITPIC algorithm). Calculated binding affinities are shown for the fitted datasets.

Article Snippet: NCoR ID1 peptide (RTHRLITLADHICQIITQDFARN) and NCoR ID2 peptide (DPASNLGLEDIIRKALMGSFDDK) were purchased with >95% from LifeTein with N-terminal amidation and C-terminal acetylation for stability and prepared as 50 mM stocks in DMSO stored at −80°C.

Techniques: Construct, Injection, Binding Assay

Journal: Science Advances

Article Title: Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

doi: 10.1126/sciadv.abc6479

Figure Lengend Snippet: Thermodynamic parameters of NCoR RID binding to REV-ERBβ LBD.

Article Snippet: NCoR ID1 peptide (RTHRLITLADHICQIITQDFARN) and NCoR ID2 peptide (DPASNLGLEDIIRKALMGSFDDK) were purchased with >95% from LifeTein with N-terminal amidation and C-terminal acetylation for stability and prepared as 50 mM stocks in DMSO stored at −80°C.

Techniques: Binding Assay

Journal: Science Advances

Article Title: Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

doi: 10.1126/sciadv.abc6479

Figure Lengend Snippet: ( A ) Differential analysis of LBD-LBD and LBD-RID cross-links in REV-ERBβ LBD + NCoR RID samples prepared in the presence or absence of heme. AF-2 cross-links involve residues K421, K439, or K576. The top left quadrant (shaded gray box) highlights cross-links significantly reduced by heme ( P adj < 0.05). ( B ) AF-2 (red) or other (pink) residues involved in cross-links significantly reduced by heme [gray box in (A)] are shown as spheres mapped to PDB 3CQV. LBD-LBD and LBD-RID cross-links involving AF-2 residues are indicated with red or black lines (all but one was significantly reduced by heme). N.S., not significant. ( C ) Differential analysis of LBD-LBD cross-links in apo (left) or heme-bound (right) REV-ERBβ LBD in the presence or absence of NCoR RID. The top right quadrant (shaded gray box) indicates cross-links significantly increased by RID binding. ( D ) LBD-LBD cross-links increased by RID in both apo and heme-bound LBD (blue) mapped to PDB 3CQV. Cross-links likely to be inter-LBD (SASD >25 Å) are indicated with dashed lines, while cross-links likely to be intra-LBD (SASD ≤25 Å) are indicated with solid lines; calculated SASDs are shown next to each line.

Article Snippet: NCoR ID1 peptide (RTHRLITLADHICQIITQDFARN) and NCoR ID2 peptide (DPASNLGLEDIIRKALMGSFDDK) were purchased with >95% from LifeTein with N-terminal amidation and C-terminal acetylation for stability and prepared as 50 mM stocks in DMSO stored at −80°C.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Defining a conformational ensemble that directs activation of PPARγ

doi: 10.1038/s41467-018-04176-x

Figure Lengend Snippet: Nuclear receptor co-regulator interaction differentiates pharmacologically distinct PPARγ ligands. TR-FRET biochemical assay shows the effect of the compounds on the interaction between PPARγ LBD and peptides derived from the a MED1 coactivator and b NCoR corepressor, plotted as TR-FRET ratio (665 nm/620 nm) vs. ligand concentration ( n = 2, standard deviation). The data shown represents technical replicates from a single experiment and the experiment was repeated four times with similar results. The window of efficacy in these data is representative of ligand-induced changes in co-regulator affinity for PPARγ. An increase in TR-FRET ratio indicates a strengthening of affinity for MED1/NCoR compared to apo while a decrease indicates a ligand-induced weakening of affinity for the co-regulator. The effect of vehicle (DMSO) is negligible (Supplementary Fig. ); furthermore, the DMSO concentration is constant across the titration both in this figure and all other TR-FRET data presented

Article Snippet: Peptides were synthesized by Lifetein LLC (Somerset, NJ, USA) for the for MED1 peptide, sequence: NTKNHPMLMNLLKDNPAQD; and the NCoR peptide, sequence: GHSFADPASNLGLEDIIRKALMG (2251–2273).

Techniques: Derivative Assay, Concentration Assay, Standard Deviation, Titration

Journal: Nature Communications

Article Title: Defining a conformational ensemble that directs activation of PPARγ

doi: 10.1038/s41467-018-04176-x

Figure Lengend Snippet: Ligand-directed helix 12 ensemble dictates PPARγ–co-regulator interaction. a Plot of mean 19 F NMR chemical shift values (PPARγ K502C -BTFA) vs. TR-FRET endpoint data for the recruitment of MED1, NCoR, and CBP peptides to PPARγ K502C -BTFA (top panels) and wt PPARγ LBD (bottom panels) for the set of 16 pharmacologically distinct synthetic PPARγ ligands and apoprotein (select ligands for CBP). Error bars are relatively small for endpoint TR-FRET values (Fig. and Supplementary Figure ) and are excluded for clarity. b Plot of mean 19 F NMR chemical shift values (PPARγ K502C -BTFA) vs. MED1, NCoR, and SMRT peptide dissociation constant ( K d ) for PPARγ K502C -BTFA (top panels) and wt PPARγ LBD (bottom panels) as measured by fluorescence polarization for a subset of the ligands in a . Linear regression fit is shown as a solid line and the correlation coefficient ( R 2 ) for the fitted line is indicated. The ligands with the highest efficacy for MED1 and NCoR recruitment for PPARγ K502C -BTFA are highlighted. Error bars represent standard deviation of two (K502C-BTFA K d and wt SMRT K d ) or three (wt MED1 and NCoR K d ) independent experiments

Article Snippet: Peptides were synthesized by Lifetein LLC (Somerset, NJ, USA) for the for MED1 peptide, sequence: NTKNHPMLMNLLKDNPAQD; and the NCoR peptide, sequence: GHSFADPASNLGLEDIIRKALMG (2251–2273).

Techniques: Fluorescence, Standard Deviation

Journal: Nature Communications

Article Title: Defining a conformational ensemble that directs activation of PPARγ

doi: 10.1038/s41467-018-04176-x

Figure Lengend Snippet: Co-regulator-binding shifts the helix 12 conformational ensemble. a Deconvoluted 19 F NMR spectra of apo PPARγ C313A,K502C -BTFA and PPARγ K502C -BTFA bound to GW1929 or T0070907 in the absence and presence of MED1 coactivator or NCoR corepressor peptides. The percent of total signal area found in the left sharp peak is shown for T0070907 and GW9662 spectra. The small sharp peak at ~−83.3 ppm is free BTFA. b Mean-weighted chemical shift values from the plots in a . c Fraction of the total peak areas in the four colored boxed regions in a , which roughly correspond to the clustered spectral regions from Fig. ; with the two middle regions corresponding to the two peaks observed for apoprotein. Agonist-bound PPARγ is changed little by MED1 binding, whereas NCoR binding changes the spectrum drastically and vice versa for inverse-agonist (T0070907)-bound PPARγ. *SMRT-induced mean chemical shift in GW1929-bound PPARγ K502C -BTFA is the same as NCoR. These experiments were performed once

Article Snippet: Peptides were synthesized by Lifetein LLC (Somerset, NJ, USA) for the for MED1 peptide, sequence: NTKNHPMLMNLLKDNPAQD; and the NCoR peptide, sequence: GHSFADPASNLGLEDIIRKALMG (2251–2273).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Defining a conformational ensemble that directs activation of PPARγ

doi: 10.1038/s41467-018-04176-x

Figure Lengend Snippet: Heterodimerization of PPARγ LBD with RXRα LBD favors coactivator binding. a 19 F NMR of PPARγ K502C -BTFA bound to T0070907 and PPARγ C313A,502C -BTFA bound to MRL24 or GW1929 or with no ligand bound was performed in the presence (orange) or absence (black) of RXRα LBD. These experiments were performed once. d The 19 F NMR signal from the MRL24 ligand. Broadening and consequent reduction in signal intensity is expected as a consequence of the increased rotational correlation time of the heterodimer complex. b , c TR-FRET was used to measure interaction between wt PPARγ LBD and MED1 or NCoR in the presence or absence of equimolar concentrations of RXRα. Error bars represent standard deviation of two technical replicates within a single experiment. The experiment was repeated twice and gave similar results each time. e Heterodimerization favors MED1 binding ( p = 0.0017) and disfavors NCoR binding ( p = 0.0076) to apo PPARγ. In addition, visual and statistical comparison of NCoR and MED1 recruitment to PPARγ LBD saturated with ligand (four highest concentrations of ligands) indicates that RXRα affects co-regulator recruitment to T0070907-bound PPARγ (NCoR, p = 0.0042; MED1, p = 0.0027) more than GW1929 (NCoR, p = 0.44; MED1, p = 0.34) or MRL24 (NCoR, p = 0.0141; MED1, p = 0.061). All p values are derived from a two-tailed t test

Article Snippet: Peptides were synthesized by Lifetein LLC (Somerset, NJ, USA) for the for MED1 peptide, sequence: NTKNHPMLMNLLKDNPAQD; and the NCoR peptide, sequence: GHSFADPASNLGLEDIIRKALMG (2251–2273).

Techniques: Binding Assay, Standard Deviation, Comparison, Derivative Assay, Two Tailed Test