ub e1  (R&D Systems)

Journal: bioRxiv

Article Title: Parp7 generates an ADP-ribosyl degron that controls negative feedback of androgen signaling

doi: 10.1101/2024.12.21.629908

Figure Lengend Snippet: DTX2 is the E3 ligase for ADP-ribosylated AR. A, Immunoblot detection of Flag-AR and AR-ADPr (by FL-AF1521) in PC3-AR cells with siRNA knockdowns (total 4-day knockdown) of the selected relevant E3 ligases (DTX1, DTX2, DTX4, HUWE1, RNF146, SPOP, TRIP12 and UBR5), treated with R1881 for 21 hr before cell harvest. The AR/TUB and AR-ADPr/AR ratios for each lane are presented below the blot. B, Immunoblot detection of Flag-AR and AR-ADPr in PC3-AR cells with siDTX2 knockdown, treated with R1881 for times indicated on the panel. The AR-ADPr/AR ratio for each lane is presented below the blot. C, Immunoblot detection of Flag-AR and AR-ADPr in PC3-AR siCTRL and siDTX2 cell extracts and AF1521 bound fraction. Cell extracts from PC3-AR siCTRL and siDTX2 cells treated with R1881 for 6 hr were combined with AF1521 beads for the enrichment of ADP-ribosylated proteins. D, Immunoblot detection of Flag-AR and AR-ADPr in PC3-AR siCTRL/siDTX2 and PC3-AR HA- PARP7 cell extracts and GSH beads bound fraction. Cell extracts from PC3-AR siCTRL/siDTX2 and PC3-AR HA-PARP7 cells treated with R1881 for 6 hr were combined with GSH beads loaded with GST- DTX2-RD or GST-DTX2-RD mut for the enrichment of proteins recognized by DTX2 DTC domain. E, Diagrams of DTX2-RD and DTX2-RD mut . Three loss of function mutations in the DTC domain of DTX2-RD mut (S568A, H582A, and H594A) are indicated with red asterisk. F, Schematic diagram of AR protein preparation as a substrate for biochemical reactions. Cell extracts from PC3-AR siDTX2 cells treated (left) or untreated (right) with R1881 for 6 hr were combined with M2 beads for immunoprecipitation. The purified protein from the preparation with R1881 treatment was used for experiments in panels G and H, and the purified protein from the preparation without R1881 treatment was used only in panel H. G, Immunoblot detection of Flag-AR and AR-ADPr from the ubiquitylation assay on AR protein prepared with siDTX2 and R1881 treatment (panel F, left). The ubiquitylated products (Ub product, red bracket) are labeled for Flag-AR and AR-ADPr detection. All reactions contained AR-ADPr (R1881 treated samples), ATP, Ub, E1 and E2. For DTX2-RD status (dropout or DTX2-RD mut ), refer to labels. H, Immunoblot detection of Flag-AR, AR-ADPr, and GST-DTX2-RD from the ubiquitylation assay on AR protein prepared with siDTX2 transfection, and with or without R1881 treatment (panel F). The ubiquitylated products (Ub product) are labeled in red for Flag-AR and AR-ADPr detection. The dropouts of the ubiquitylation assay components (Ub, E1, E2, and 30°C incubation) are indicated on the labels. The T7-Ubiquitin (T7-Ub) was detected by Ponceau staining. Lane numbers are indicated below the blot. I, Bar plots showing the results of an RT-qPCR experiment in PC3-AR siCTRL/siDTX2 cells untreated (grey), treated with R1881 (purple), and cotreated with R1881 and RBN2397 (blue). The y-axis represents the relative expression normalized to the GUS housekeeping gene, and the x-axis represents the siRNA used. The p-values from the Welch’s t-test for comparisons between corresponding conditions in siCTRL and siDTX2 are indicated on the plots. Error bars represent standard deviation (n = 3; n represents number of biological replicates).

Article Snippet: Ubiquitylation assays were performed at 30°C for 30 min with 1 mM ATP, 100 g/ml Ub (T7-Ub or bovine Ub (Sigma U6253)), 5 g/ml each of UB E1 (R&D Systems E-304) and UB E2 (His-UbcH5C, R&D Systems E2-627), and 20 g/ml GST-DTX2 RD in the buffer E (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM DTT, 1 µg/ml each of A/L/P and 0.1 mg/ml BSA).

Techniques: Western Blot, Knockdown, Immunoprecipitation, Purification, Ubiquitin Assay, Labeling, Transfection, Incubation, Staining, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Parp7 generates an ADP-ribosyl degron that controls negative feedback of androgen signaling

doi: 10.1101/2024.12.21.629908

Figure Lengend Snippet: DTX2 conjugates ubiquitin to AR through ADP-ribose A, Immunoblot detection of Fluorescein (FITC) and T7-Ubiquitin (T7-Ub) from the ubiquitylation assay on FITC-AR(C284) or FITC-AR(C284 ADPr ) peptides. The labels indicate from the top: the substrate used (FITC-AR(C284) or FITC-AR(C284 ADPr ) peptides), pre-ubiquitylation assay treatments (NUDT16), the ubiquitylation assay (all reactions contained ATP, T7-Ub, E1 and E2, for DTX2-RD dropout, refer to labels), and the post- ubiquitylation assay treatments (NUDT16 or Mg 2+ buffer). Lane numbers are indicated below the blot. B, Schematic diagram representing FITC-AR(C284 ADPr ) peptide conjugated to ubiquitin (Ub). Indicated with red scissors are bonds within the ADP-ribose structure cleaved by NUDT16 and USP2. C, Schematic of the ubiquitylation assay workflow for panels D and E. D, Immunoblot detection of Flag-AR and AR-ADPr (by FL-AF1521) from the ubiquitylation assay on AR protein prepared with siDTX2 transfection and R1881 treatment (refer to panel 6f for sample preparation workflow). The ubiquitylated products (Ub product) are labeled in red for Flag-AR and AR-ADPr detection. The labels separated by black lines indicate sequential steps from top to bottom. From the top, the labels indicate the substrate used (AR-ADPr), pre-ubiquitylation assay treatments (NUDT16), the ubiquitylation assay, and the post-ubiquitylation assay treatments (USP2-CD, NUDT16, or Mg 2+ buffer). Lane numbers are indicated below the blot. E, Immunoblot detection of Flag-AR and AR-ADPr from the ubiquitylation assay on AR protein prepared with siDTX2 and R1881 treatment (refer to panel 6f for sample preparation workflow). The ubiquitylated products (Ub product) are labeled in red for Flag-AR and AR-ADPr detection. The labels separated by black lines indicate sequential steps from top to bottom. From the top, the labels indicate the substrate used (AR-ADPr), pre-ubiquitylation assay treatments (NUDT16), the ubiquitylation assay, and the post- ubiquitylation assay treatments (USP2-CD, NUDT16, or Mg 2+ buffer). Lane numbers are indicated below the blot. F, Scatter plots depicting the correlation between PARP7 (left) or DTX2 (right) mRNA expression and the response to androgen pathway activity calculated by PARADIGM in primary prostate cancer patients from TCGA-PRAD cohort. Each dot represents one patient (n = 478, n represents number of patients). Pearson correlation coefficients and corresponding p-values are indicated on the plots. G, Kaplan-Meier plot depicting progression-free interval (PFI) in primary prostate cancer patients from the TCGA-PRAD cohort, stratified by PARP7 expression levels. The red line represents patients with high PARP7 expression (top 25%), and the green line represents patients with low PARP7 expression (bottom 25%). The X-axis represents time (days), and the Y-axis represents the progression-free interval probability. The interval distributions were compared using the log-rank test, with the p-value indicating statistical significance. Dotted lines represent the 95% confidence interval.

Article Snippet: Ubiquitylation assays were performed at 30°C for 30 min with 1 mM ATP, 100 g/ml Ub (T7-Ub or bovine Ub (Sigma U6253)), 5 g/ml each of UB E1 (R&D Systems E-304) and UB E2 (His-UbcH5C, R&D Systems E2-627), and 20 g/ml GST-DTX2 RD in the buffer E (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM DTT, 1 µg/ml each of A/L/P and 0.1 mg/ml BSA).

Techniques: Western Blot, Ubiquitin Assay, Transfection, Sample Prep, Labeling, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Bacterial deamidases modulate ubiquitin structure and dynamics to dysregulate ubiquitin signaling

doi: 10.1101/2023.05.22.541748

Figure Lengend Snippet: Deamidation induces the formation of new salt bridges in ubiquitin. (A) In-vitro ubiquitination reaction was performed using Ubc13 as the E2, RNF38 RING as the E3, and E40Ub or Q40Ub for 10 min. Mms2 was used as a co-factor in the reaction. The –ve lane is the same reaction without ATP. (B) The same assay as in (A) is repeated with the E2 Ubch5b. (C) An overlay of 1 H- 15 N HSQCs of Q40Ub and E40Ub. (D) Chemical Shift Perturbations (CSPs) in amide resonances between Q40Ub and E40Ub are plotted against the Ub residue numbers. Residues in the c-terminal tail and around E40 show changes in the chemical environment. The secondary structure of Ub is shown above the plot. The broken black line is Standard Deviation (SD), and the red line is 2*SD. (E) CSPs plotted for Arginine Nε-Hε resonances between Q40Ub and E40Ub. (F) A superposition of crystal structures of Q40Ub (PDB id: 1UBQ) and E40Ub (this work). (G) A zoomed panel shows the electron density and packing of R74 between D39 and E40. (H) The new salt bridge formed between R74 and E40 is shown as black lines. A new hydrogen bond is also formed between E40 and the R72 backbone. The length of salt bridges and hydrogen bonds are mentioned. (I) Salt-bridge occupancies were calculated from two independent MD runs of E40Ub using a 0.5 nm cutoff.

Article Snippet: To monitor chemical shift perturbations (CSPs) and change in intensities of Ub/E40Ub peaks upon E1 binding, 180 µL of equimolar (65 µM) 15 N/D 2 O labeled Ub/E40Ub, and E1 was prepared in 25 mM Tris-HCl, 50 mM NaCl (pH 7.5), 10% D 2 O. TROSY spectra were collected in an 800 MHz Bruker Avance III HD spectrometer AT 298K.

Techniques: In Vitro, Standard Deviation

Journal: bioRxiv

Article Title: Bacterial deamidases modulate ubiquitin structure and dynamics to dysregulate ubiquitin signaling

doi: 10.1101/2023.05.22.541748

Figure Lengend Snippet: Deamidation perturbs the conformational dynamics and non-covalent interactions of Ub. (A) The chemical shift perturbation (CSP) in Ub, when bound to the p62-UBA domain. (B) The CSPs are plotted for the p62-UBA domain when bound to Ub. (C) The CSPs of Q40Ub and E40Ub when titrated with various concentrations of p62-UBA. (D) The dissociation constants of UBA/Ub complexes are obtained by fitting the titrations in (C). (E) The structure of the p62-UBA(white)/Q40Ub(orange) complex is shown. The E40Ub (purple) is superimposed on the Q40Ub structure. (F) The intermolecular salt bridge between D45 in UBA and R74 in Q40Ub is highlighted in black. The R74 in E40Ub is orientated in the opposite direction due to the intramolecular salt bridge with D39. (G) The intermolecular salt bridges between D45 in UBA and R72 in Q40Ub are highlighted. The C-terminal tail in E40Ub is distant from the UBA domain due to the hydrogen bond with E40. The R72 is orientated away from a potential intermolecular salt bridge with D45. (H) 15 N relaxation dispersion for the amide of the ubiquitin residue I41 at 80 MHz in the presence of 0:05 mM and 0:5 mM of p62-UBA domain (blue and red data point). (I) The obtained exchange rates kex of the ubiquitin residue 41 (black data points with error bars) decrease with increasing p62-UBA concentration, indicating conformational selection. The gray lines with shaded error regions result from fits of the k ex equations of the two-state and conformational-selection binding mechanism. The red line denotes the induced fit model. (J) and (K) are the order parameters (S 2 ) and spin-spin relaxation rates (R 2 ), respectively, of the C-terminal tail residues in Q40Ub and E40Ub. The same for complete protein is provided in Figure S4. (L) The 15 N relaxation dispersion data (R 2eff against ν CPMG ) are plotted for a few selected residues in Q40Ub (orange) and E40Ub (purple). (M) and (N) plots the k ex and Θ (p A p B Δω 2 ), respectively, for the residues with high R ex .

Article Snippet: To monitor chemical shift perturbations (CSPs) and change in intensities of Ub/E40Ub peaks upon E1 binding, 180 µL of equimolar (65 µM) 15 N/D 2 O labeled Ub/E40Ub, and E1 was prepared in 25 mM Tris-HCl, 50 mM NaCl (pH 7.5), 10% D 2 O. TROSY spectra were collected in an 800 MHz Bruker Avance III HD spectrometer AT 298K.

Techniques: Concentration Assay, Selection, Binding Assay

Journal: bioRxiv

Article Title: Bacterial deamidases modulate ubiquitin structure and dynamics to dysregulate ubiquitin signaling

doi: 10.1101/2023.05.22.541748

Figure Lengend Snippet: Deamidation retards the rate of E1 activation. (A) The ratio of Ub backbone amide resonance intensities (I complex /I free ) in the Q40Ub/E1 and E40Ub/E1 non-covalent complex measured by 15 N-TROSY-HSQC is plotted against the Ub residue numbers. The Ub molecules were isotopically labeled with 2 H and 15 N. I complex is the intensity of Ub resonances in the Ub/E1 complex and I free if that of free Ub. Extended data in Supp fig XX. (B) The CSPs in Ub in the Q40Ub/E1 or E40Ub/E1 complexes are plotted against Ub residue numbers. (C) Change in the fluorescence anisotropy of Q40Ub or E40Ub plotted against increasing E1 concentration is plotted, which yielded the dissociation constant of the E1/Ub complex. The Ub molecules were labeled with Alexa488. (D) ESI-MS analysis of the rate of E1~Ub conjugate formation in Q40Ub and E40Ub. Extended data in Fig S6. (E) The fraction of E1-conjugated Q40Ub and E40Ub is plotted against time. The R72A-Ub is plotted as a control. (F) Rate of E1~Ub to E2~Ub trans-thiolation is plotted as the fraction (E2~Ub)/((E1~Ub) +(E2~Ub)) against time. The Ub molecules were labeled with Alexa488. Extended data in Supp fig XX. (G) A plot of E2~Q40Ub and E2~E40Ub conjugation against time for the E2 UbcH5b. (J) The E2~Q40Ub and E2~E40Ub formation rates were measured for the E2s UbcH5b, Ubc13, and UbE2B.

Article Snippet: To monitor chemical shift perturbations (CSPs) and change in intensities of Ub/E40Ub peaks upon E1 binding, 180 µL of equimolar (65 µM) 15 N/D 2 O labeled Ub/E40Ub, and E1 was prepared in 25 mM Tris-HCl, 50 mM NaCl (pH 7.5), 10% D 2 O. TROSY spectra were collected in an 800 MHz Bruker Avance III HD spectrometer AT 298K.

Techniques: Activation Assay, Labeling, Fluorescence, Concentration Assay, Conjugation Assay

Journal: bioRxiv

Article Title: Bacterial deamidases modulate ubiquitin structure and dynamics to dysregulate ubiquitin signaling

doi: 10.1101/2023.05.22.541748

Figure Lengend Snippet: Deamidation perturbs the E2~Ub conformational equilibrium. (A) In-vitro ubiquitination reaction was performed using Ubc13 as the E2 and E40Ub or Q40Ub for 20 min. Mms2 was used as a co-factor in the reaction. The –ve lane is the same reaction without ATP. (B) Same as in (A) using Ube2K as the E2. (C) The model of deamidation induced perturbation of the E2~Ub dynamics. (D) Mean force-time profiles were obtained from Steered MD simulations of Ubc13~Ub and various Ub mutants. (E) Mean F max and unbinding work (W) for the dissociation of Ub from the Ubc13~Ub closed-state. One standard error of the mean is indicated in the brackets. (F) Combined probability distribution of Ubc13/Ub intermolecular angle in the Ubc13~Q40Ub and Ubc13~E40Ub trajectories. (G) 2D plots showing the correlation between intramolecular salt-bridge and Ubc13/E40Ub angles. The higher values of angles correlate with the formation of E40 salt bridges. (H) Representative structures of high probability state in the E2~Q40Ub and E2~E40Ub conjugates. The hydrophobic patch in Ub and the central α-helix α2 are colored yellow. (I) The change in RMSD of Q40Ub (black) and E40Ub (red) is plotted against time from targeted MD simulations of E2~Ub from open to closed conformation. The RMSD is calculated relative to Ub in the E2~Ub closed conformation. (J) The angle between Ubc13 helix II and Ub helix I from the same trajectory is plotted against time. (K) The elution profile of E2 and E2~Ub in size-exclusion chromatography.

Article Snippet: To monitor chemical shift perturbations (CSPs) and change in intensities of Ub/E40Ub peaks upon E1 binding, 180 µL of equimolar (65 µM) 15 N/D 2 O labeled Ub/E40Ub, and E1 was prepared in 25 mM Tris-HCl, 50 mM NaCl (pH 7.5), 10% D 2 O. TROSY spectra were collected in an 800 MHz Bruker Avance III HD spectrometer AT 298K.

Techniques: In Vitro, Size-exclusion Chromatography

Journal: bioRxiv

Article Title: Bacterial deamidases modulate ubiquitin structure and dynamics to dysregulate ubiquitin signaling

doi: 10.1101/2023.05.22.541748

Figure Lengend Snippet: The distorted conformation of E2~E40Ub fails to bind RING domains. (A) The difference in CSPs (ΔCSPs) of Ubc13 when conjugated to Q40Ub and E40Ub. ΔCSP = CSP(Ubc13~Q40Ub) – CSP(Ubc13~E40Ub). (B) Same as in (A) is plotted for Ub. (C) The population of open and closed states calculated for the Ubc13~Ub/RNF38 RING complex is calculated by the model provided in Figure S16. (D) Ubc13~Q40Ub and Ubc13~E40Ub model structures generated using NMR CSPs by HADDOCK show differences in Ub orientation between the open and closed conformation. (E) A Ubc13~Q40Ub/RNF38 RING model suggests multiple interactions between Ub and RNF38 RING that stabilize the closed conformation. (F) The Ub/RING interactions are absent in a similar Ubc13~E40Ub/RNF38 RING model. (G) CSPs in Ub are plotted for the Ubc13~Q40Ub/RNF38 RING complex. The dark blue and light blue dashed lines correspond to Mean+2*SD. The residues in the UB/RNF38 RING interface observed in (D) are marked with asterisks. (H) CSPs in Ub are plotted for the Ubc13~E40Ub/RNF38 RING complex. The lack of high CSP values indicates a loss of interaction between E40Ub and the RING domain. (I) Pull-down assay with GST-RNF4 RING and Ubc13~Ub shows loss of RING binding upon deamidation.

Article Snippet: To monitor chemical shift perturbations (CSPs) and change in intensities of Ub/E40Ub peaks upon E1 binding, 180 µL of equimolar (65 µM) 15 N/D 2 O labeled Ub/E40Ub, and E1 was prepared in 25 mM Tris-HCl, 50 mM NaCl (pH 7.5), 10% D 2 O. TROSY spectra were collected in an 800 MHz Bruker Avance III HD spectrometer AT 298K.

Techniques: Generated, Pull Down Assay, Binding Assay

Journal: bioRxiv

Article Title: Bacterial deamidases modulate ubiquitin structure and dynamics to dysregulate ubiquitin signaling

doi: 10.1101/2023.05.22.541748

Figure Lengend Snippet: Deamidation induces the formation of new salt bridges in ubiquitin. (A) In-vitro ubiquitination reaction was performed using Ubc13 as the E2, RNF38 RING as the E3, and E40Ub or Q40Ub for 10 min. Mms2 was used as a co-factor in the reaction. The –ve lane is the same reaction without ATP. (B) The same assay as in (A) is repeated with the E2 Ubch5b. (C) An overlay of 1 H- 15 N HSQCs of Q40Ub and E40Ub. (D) Chemical Shift Perturbations (CSPs) in amide resonances between Q40Ub and E40Ub are plotted against the Ub residue numbers. Residues in the c-terminal tail and around E40 show changes in the chemical environment. The secondary structure of Ub is shown above the plot. The broken black line is Standard Deviation (SD), and the red line is 2*SD. (E) CSPs plotted for Arginine Nε-Hε resonances between Q40Ub and E40Ub. (F) A superposition of crystal structures of Q40Ub (PDB id: 1UBQ) and E40Ub (this work). (G) A zoomed panel shows the electron density and packing of R74 between D39 and E40. (H) The new salt bridge formed between R74 and E40 is shown as black lines. A new hydrogen bond is also formed between E40 and the R72 backbone. The length of salt bridges and hydrogen bonds are mentioned. (I) Salt-bridge occupancies were calculated from two independent MD runs of E40Ub using a 0.5 nm cutoff.

Article Snippet: To monitor chemical shift perturbations (CSPs) and change in intensities of Ub/E40Ub peaks upon E1 binding, 180 µL of equimolar (65 µM) 15 N/D 2 O labeled Ub/E40Ub, and E1 was prepared in 25 mM Tris-HCl, 50 mM NaCl (pH 7.5), 10% D 2 O. TROSY spectra were collected in an 800 MHz Bruker Avance III HD spectrometer AT 298K.

Techniques: In Vitro, Standard Deviation

Journal: bioRxiv

Article Title: Bacterial deamidases modulate ubiquitin structure and dynamics to dysregulate ubiquitin signaling

doi: 10.1101/2023.05.22.541748

Figure Lengend Snippet: Deamidation perturbs the conformational dynamics and non-covalent interactions of Ub. (A) The chemical shift perturbation (CSP) in Ub, when bound to the p62-UBA domain. (B) The CSPs are plotted for the p62-UBA domain when bound to Ub. (C) The CSPs of Q40Ub and E40Ub when titrated with various concentrations of p62-UBA. (D) The dissociation constants of UBA/Ub complexes are obtained by fitting the titrations in (C). (E) The structure of the p62-UBA(white)/Q40Ub(orange) complex is shown. The E40Ub (purple) is superimposed on the Q40Ub structure. (F) The intermolecular salt bridge between D45 in UBA and R74 in Q40Ub is highlighted in black. The R74 in E40Ub is orientated in the opposite direction due to the intramolecular salt bridge with D39. (G) The intermolecular salt bridges between D45 in UBA and R72 in Q40Ub are highlighted. The C-terminal tail in E40Ub is distant from the UBA domain due to the hydrogen bond with E40. The R72 is orientated away from a potential intermolecular salt bridge with D45. (H) 15 N relaxation dispersion for the amide of the ubiquitin residue I41 at 80 MHz in the presence of 0:05 mM and 0:5 mM of p62-UBA domain (blue and red data point). (I) The obtained exchange rates kex of the ubiquitin residue 41 (black data points with error bars) decrease with increasing p62-UBA concentration, indicating conformational selection. The gray lines with shaded error regions result from fits of the k ex equations of the two-state and conformational-selection binding mechanism. The red line denotes the induced fit model. (J) and (K) are the order parameters (S 2 ) and spin-spin relaxation rates (R 2 ), respectively, of the C-terminal tail residues in Q40Ub and E40Ub. The same for complete protein is provided in Figure S4. (L) The 15 N relaxation dispersion data (R 2eff against ν CPMG ) are plotted for a few selected residues in Q40Ub (orange) and E40Ub (purple). (M) and (N) plots the k ex and Θ (p A p B Δω 2 ), respectively, for the residues with high R ex .

Article Snippet: To monitor chemical shift perturbations (CSPs) and change in intensities of Ub/E40Ub peaks upon E1 binding, 180 µL of equimolar (65 µM) 15 N/D 2 O labeled Ub/E40Ub, and E1 was prepared in 25 mM Tris-HCl, 50 mM NaCl (pH 7.5), 10% D 2 O. TROSY spectra were collected in an 800 MHz Bruker Avance III HD spectrometer AT 298K.

Techniques: Concentration Assay, Selection, Binding Assay

Journal: bioRxiv

Article Title: Bacterial deamidases modulate ubiquitin structure and dynamics to dysregulate ubiquitin signaling

doi: 10.1101/2023.05.22.541748

Figure Lengend Snippet: Deamidation retards the rate of E1 activation. (A) The ratio of Ub backbone amide resonance intensities (I complex /I free ) in the Q40Ub/E1 and E40Ub/E1 non-covalent complex measured by 15 N-TROSY-HSQC is plotted against the Ub residue numbers. The Ub molecules were isotopically labeled with 2 H and 15 N. I complex is the intensity of Ub resonances in the Ub/E1 complex and I free if that of free Ub. Extended data in Supp fig XX. (B) The CSPs in Ub in the Q40Ub/E1 or E40Ub/E1 complexes are plotted against Ub residue numbers. (C) Change in the fluorescence anisotropy of Q40Ub or E40Ub plotted against increasing E1 concentration is plotted, which yielded the dissociation constant of the E1/Ub complex. The Ub molecules were labeled with Alexa488. (D) ESI-MS analysis of the rate of E1~Ub conjugate formation in Q40Ub and E40Ub. Extended data in Fig S6. (E) The fraction of E1-conjugated Q40Ub and E40Ub is plotted against time. The R72A-Ub is plotted as a control. (F) Rate of E1~Ub to E2~Ub trans-thiolation is plotted as the fraction (E2~Ub)/((E1~Ub) +(E2~Ub)) against time. The Ub molecules were labeled with Alexa488. Extended data in Supp fig XX. (G) A plot of E2~Q40Ub and E2~E40Ub conjugation against time for the E2 UbcH5b. (J) The E2~Q40Ub and E2~E40Ub formation rates were measured for the E2s UbcH5b, Ubc13, and UbE2B.

Article Snippet: To monitor chemical shift perturbations (CSPs) and change in intensities of Ub/E40Ub peaks upon E1 binding, 180 µL of equimolar (65 µM) 15 N/D 2 O labeled Ub/E40Ub, and E1 was prepared in 25 mM Tris-HCl, 50 mM NaCl (pH 7.5), 10% D 2 O. TROSY spectra were collected in an 800 MHz Bruker Avance III HD spectrometer AT 298K.

Techniques: Activation Assay, Labeling, Fluorescence, Concentration Assay, Conjugation Assay

Journal: bioRxiv

Article Title: Bacterial deamidases modulate ubiquitin structure and dynamics to dysregulate ubiquitin signaling

doi: 10.1101/2023.05.22.541748

Figure Lengend Snippet: Deamidation perturbs the E2~Ub conformational equilibrium. (A) In-vitro ubiquitination reaction was performed using Ubc13 as the E2 and E40Ub or Q40Ub for 20 min. Mms2 was used as a co-factor in the reaction. The –ve lane is the same reaction without ATP. (B) Same as in (A) using Ube2K as the E2. (C) The model of deamidation induced perturbation of the E2~Ub dynamics. (D) Mean force-time profiles were obtained from Steered MD simulations of Ubc13~Ub and various Ub mutants. (E) Mean F max and unbinding work (W) for the dissociation of Ub from the Ubc13~Ub closed-state. One standard error of the mean is indicated in the brackets. (F) Combined probability distribution of Ubc13/Ub intermolecular angle in the Ubc13~Q40Ub and Ubc13~E40Ub trajectories. (G) 2D plots showing the correlation between intramolecular salt-bridge and Ubc13/E40Ub angles. The higher values of angles correlate with the formation of E40 salt bridges. (H) Representative structures of high probability state in the E2~Q40Ub and E2~E40Ub conjugates. The hydrophobic patch in Ub and the central α-helix α2 are colored yellow. (I) The change in RMSD of Q40Ub (black) and E40Ub (red) is plotted against time from targeted MD simulations of E2~Ub from open to closed conformation. The RMSD is calculated relative to Ub in the E2~Ub closed conformation. (J) The angle between Ubc13 helix II and Ub helix I from the same trajectory is plotted against time. (K) The elution profile of E2 and E2~Ub in size-exclusion chromatography.

Article Snippet: To monitor chemical shift perturbations (CSPs) and change in intensities of Ub/E40Ub peaks upon E1 binding, 180 µL of equimolar (65 µM) 15 N/D 2 O labeled Ub/E40Ub, and E1 was prepared in 25 mM Tris-HCl, 50 mM NaCl (pH 7.5), 10% D 2 O. TROSY spectra were collected in an 800 MHz Bruker Avance III HD spectrometer AT 298K.

Techniques: In Vitro, Size-exclusion Chromatography

Journal: bioRxiv

Article Title: Bacterial deamidases modulate ubiquitin structure and dynamics to dysregulate ubiquitin signaling

doi: 10.1101/2023.05.22.541748

Figure Lengend Snippet: The distorted conformation of E2~E40Ub fails to bind RING domains. (A) The difference in CSPs (ΔCSPs) of Ubc13 when conjugated to Q40Ub and E40Ub. ΔCSP = CSP(Ubc13~Q40Ub) – CSP(Ubc13~E40Ub). (B) Same as in (A) is plotted for Ub. (C) The population of open and closed states calculated for the Ubc13~Ub/RNF38 RING complex is calculated by the model provided in Figure S16. (D) Ubc13~Q40Ub and Ubc13~E40Ub model structures generated using NMR CSPs by HADDOCK show differences in Ub orientation between the open and closed conformation. (E) A Ubc13~Q40Ub/RNF38 RING model suggests multiple interactions between Ub and RNF38 RING that stabilize the closed conformation. (F) The Ub/RING interactions are absent in a similar Ubc13~E40Ub/RNF38 RING model. (G) CSPs in Ub are plotted for the Ubc13~Q40Ub/RNF38 RING complex. The dark blue and light blue dashed lines correspond to Mean+2*SD. The residues in the UB/RNF38 RING interface observed in (D) are marked with asterisks. (H) CSPs in Ub are plotted for the Ubc13~E40Ub/RNF38 RING complex. The lack of high CSP values indicates a loss of interaction between E40Ub and the RING domain. (I) Pull-down assay with GST-RNF4 RING and Ubc13~Ub shows loss of RING binding upon deamidation.

Article Snippet: To monitor chemical shift perturbations (CSPs) and change in intensities of Ub/E40Ub peaks upon E1 binding, 180 µL of equimolar (65 µM) 15 N/D 2 O labeled Ub/E40Ub, and E1 was prepared in 25 mM Tris-HCl, 50 mM NaCl (pH 7.5), 10% D 2 O. TROSY spectra were collected in an 800 MHz Bruker Avance III HD spectrometer AT 298K.

Techniques: Generated, Pull Down Assay, Binding Assay