EMBO/soda-nel huggingface dataset:
Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes (2021)
Figure 5

Figure 5 - UBE2SHTH interacts with the donor Ub, suggesting that APC2 can prevent this conformation to facilitate the "closed" conformation.

A Molecular dynamics simulation of UBE2S (green)~Ub (yellow) reveals electrostatic interactions between the positive surface of Ub and UBE2S E139 (cutout). Rosetta model of APC2 (gray mesh, from PBD: 5L9T) docked on UBE2S~Ub would sterically clash with this extended conformation (Brown et al., 2016).

B Reducing the positive charge of *Ub reduces the autoubiquitination of the E139R-substituted UBE2S variant, monitored by fluorescent scanning of an SDS-PAGE gel.

C HSQC spectra displaying chemical shift perturbations and linewidth changes in 15N-labeled UBE2SUBC WT apo (black) or in complex with Ub (red) and UBE2SUBC ER/ER apo (blue) or in complex with Ub (green).

D Plot of 15N-labeled UBE2SUBC WT and ER/ER 1H linewidth changes in the presence of Ub revealing the ER/ER variant has a more dynamic active-site cysteine and HTH, but a more stabilized crossover helix, indicative of the "closed" state. Shown are the best-fit values and standard deviations (SD) estimated from the covariance matrix analysis of NMR spectra with n = 2048 measurement points for 16 ppm in the 1H dimension.

E Structural view of UBE2S C118 interacting with I44 of Ub in the "closed" conformation of UBE2S~Ub. UBE2S (PDB: 1ZDN) was aligned to a "closed" form of UBE2D~Ub (PDB: 4AUQ) (Dou et al., 2012; Sheng et al., 2012).

F C118A substitutions decrease UBE2S autoubiquitination, measured by fluorescent scanning of SDS-PAGE gels, indicating the significance of the "closed" formation of the UBE2S~Ub for activity.

G Rosetta modeling of UBE2S~Ub, in the absence of APC2, reveals multiple "open" conformations that would sterically clash with a docked Rosetta model of the APC2 (gray mesh, PDB: 5L9T) (Brown et al., 2016). Cutout showing multiple possible interactions between UBE2S and Ub.

H Previous UBE2S~Ub crystal structure would also be prevented by the binding of APC2 (gray mesh, PDB: 5L9T) (Brown et al., 2016; Lorenz et al., 2016).
Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes (2021)
Figure 6

Figure 6 - E2HTH substitutions differentially impact substrate ubiquitination by the E3 APC/C.

A, B Schematic of UBE2S~Ub (A) and UBE2C~Ub interactions (B) with the APC/C. UBE2S~Ub uses its C-terminal extension to bind to a groove formed by APC2 and APC4. The UBE2SHTH interacts with APC2 and the APC11 RING domain positions the substrate-linked Ub for chain elongation. UBE2C~Ub is positioned by the APC11RING and APC2 winged-helix B (WHB) domains during substrate ubiquitination (Brown et al., 2015; Brown et al., 2016).

C Securin ubiquitination by APC/C and either UBE2C WT or K157E, resolved by SDS-PAGE and fluorescent scanning. Quantification of 4 or more ubiquitins added on the substrate (right) are calculated as a fraction of total ubiquitination and analyzed by an unpaired Welch's t-test (**** p ≤ 0.0001, n=5 independent, technical replicates). Error bars: SEM.

D Similar to C, Ub-securin ubiquitination by APC/C and either UBE2D2 WT or charge-swap variants. Quantification of 4 or more ubiquitins (right) added on the substrate are calculated as a fraction of total ubiquitination and analyzed by one-way ANOVA (**** p ≤ 0.0001, n=4 independent, technical replicates). Error bars: SEM.

E Representative fluorescent scan of an SDS-PAGE gel of APC/C-dependent polyubiquitination of Ub-CyclinB* by a panel of UBE2SHTH charge-swap variants. Quantification in Fig EV5C.

F The recombinant UBE2S ER/ER variant increases Cyclin B degradation in G1 extracts compared to UBE2S wild-type (WT), monitored by immunoblot. G1 extracts were prepared from HeLa S3 cells treated with either control firefly luciferase siRNA (siFF) or two UBE2S siRNAs (siUBE2S).
Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes (2021)
Figure 7

Figure 7 - UBE2SHTH substitutions partially rescue the defects in Ub chain elongation created by disrupting the UBE2S-APC2 interaction.

A Structural view of UBE2SHTH (green) interacting with APC2 for diUb synthesis (Brown et al., 2016). Residues of interest at the UBE2SHTH-APC2 interface are indicated as red spheres.

B Representative fluorescent scan of an SDS-PAGE gel (left) and its quantification (right) where the charged-swapped variants of the UBE2SHTH enhance substrate ubiquitination of APC/Cs harboring APC2 substitutions. Statistical significance was assessed by two-way ANOVA (* p ≤ 0.05, *** p ≤ 0.001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to the negative control substrate band from reactions lacking an E2.

C APC/C-UBE2S-dependent diUb synthesis was monitored by fluorescent scanning (left), quantitated (right), and assessed for statistical significance by two-way ANOVA (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates), demonstrating that the UBE2SHTH substitutions rescue the defects in diUb synthesis created by mutating APC2. Error bars: SEM. Data normalized to WT APC/C-UBE2S diUb reactions.

D APC/C Platform-dependent activation of UBE2S-mediated diUb synthesis was monitored by fluorescent scanning (left), quantitated (right), and assessed for statistical significance by two-way ANOVA (* p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to WT APC/C Platform-UBE2S diUb reactions.

E Similar to B, the role of the "closed" conformation in UBE2S-dependent Ub chain elongation of the APC/C substrate Ub-CycB*, monitored by the incorporation of C118A-substituted UBE2S variants (top). Quantification of ubiquitinated Ub-CycB* was assessed (bottom) and statistical significance by one-way ANOVA between C118A and WT, C118A/E139R, and C118A/E143R (** p ≤ 0.01, **** p ≤ 0.0001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to WT UBE2S 4+ Ub additions.

EXPANDED VIEW FIGURE LEGENDS
Oligomeric interactions maintain active-site structure in a non-cooperative enzyme family (2022)
Figure 4

Figure 4. Kinetic analyses of NADPH-dependent reduction of prochiral ketones by SCR and SCR-his6 .

(A) Michaelis-Menten analysis of the reduction of 2-hydroxyacetophenone (2-HAP).

(B) Comparison of kcat values obtained from Michaelis-Menten analyses of the reduction of four different substrates. Their chemical structures are show in the Appendix Fig S6A. The initial velocities (V0/[Etot]) and turnover rates (kcat) are given for the individual subunits (i.e., normalized to subunit not oligomer concentration). Enzyme assays were conducted at 30 ˚C in 100 mM sodium phosphate (pH 6.0) using 500 µM NADPH and 100 nM SCR or 1 µM SCR-his6. The specific activity of SCR does not change when enzyme subunit concentration is varied over a 10-fold range from 10-100 nM (Appendix Fig S11). Enzyme activity at each substrate concentration was assayed at least three and usually four times. The data points show the average values for these technical replicates, while the error bars show the standard deviations.
Oligomeric interactions maintain active-site structure in a non-cooperative enzyme family (2022)
Figure 6

Figure 6. Enzymological and structural characterization of the SCR-SCR2 heterotetramer.

(A) Analytical gel-filtration of purified his8-MBP-SCR2 (black), strep-SCR (yellow), and his8-MBP-SCR2/strep-SCR on a Superdex 200 Increase column at 20 ˚C in 150 mM NaCl, 50 mM Tris-HCl, pH 8.0.

(B) Michaelis-Menten kinetic analyses of NADPH-dependent reduction of 2-hydroxyacetophenone and methyl acetoacetate at 30 ˚C in 100 mM sodium phosphate, pH 6.0. The turnover rate (V0/[Etot]) is given for the individual subunits (as in Fig 4). Enzyme activity at each substrate concentration was assayed at least three and usually four times. The data points show the average values for these technical replicates, while the error bars show the standard deviations.

(C) Sequence alignment of SCR and SCR2 from residue 81 to residue 279. Residues 1-80 are identical. The cyan stars highlight substitutions of sidechains exposed in the active site cavity.

(D) 2Fo‑Fc (blue) and Fo‑Fc (positive/negative in green/red, respectively) electron density maps for residues 268-272 in the X-ray crystal structure of the NADPH complex of the SCR-SCR2 heterotetramer refined using superimposed dimers or dimer as coordinate models (Appendix Table S5).

(E) Superposition of subunits of SCR (yellow) and SCR2 (green) in the heterotetramer crystal structure. All residues differing between the two paralogs are shown in space-filling representation in cyan in the active site and blue elsewhere. The bound NADPH and the C-terminal residue in each subunit are shown in gray and black space-filling representations, respectively.

(F) The complete heterotetramer crystal from the SCR-SCR2 structure colored the same way.

(G) Superposition of the SCR-SCR2 heterotetramer colored the same way with the SCR homotetramer colored as in Fig 3.
Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E (2016)
Figure 1

Figure 1. UV-crosslinking of RNase E reveals binding sites transcriptome-wide.

A. Schematic of CLASH protocol for purification of RNA-RNA interactions. RNAs were UV-crosslinked to RNase E-HTF in vivo and purified using M2 anti-FLAG resin. RNAs were trimmed using RNase A/T1 and further purified under denaturing conditions. RNA linkers were ligated to the immobilized RNA-RNase E complexes. Duplexed RNAs may be ligated into a single contiguous molecule (left, CLASH) that gives information on RNA-RNA interaction occurring on RNase E. The remaining single RNAs reveal the site of RNase E binding within the transcriptome. Linker ligated RNA-RNase E complexes were size selected by SDS-PAGE and RNAs recovered for library preparation and sequencing.

B. The 5’UTR of rne is bound by RNase E and non-genomically encoded A-tails are maximally recovered -9nt from the rne start codon. Known stem loop strucutres (HP1-3) and the ribosomal binding site (RBS) are shaded grey.

C. RNase E binding and oligoadenylation of the pdlB-yigL dicistronic transcript. The reported RNase E cleavage site (red dashed line) and SgrS binding site (grey shading) are indicated.

D. Length of non-genomically encoded oligo(A) tails recovered from RNase E bound reads.

E. Position of Hfq binding and oliogadenylation relative to RNase E binding peaks. The cumulative position of Hfq and oligoadenylation peaks were determined relative to RNase E binding peaks for 672 RNase E binding sites that were within 1kb of an Hfq binding peak. A detailed description of the data processing is presented in the Appended Supplementary Methods.
Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E (2016)
Figure 2

Figure 2. Relative position, RNA class, and base pairing strength of small RNA hybrids recovered by RNase E CLASH.

A. In silico folding of hybrid reads using the UNAfold suite of tools was used to predict base pairing and interaction strength between hybrid read halves. Reads mapping to the interaction between MicA and ompA mRNA are shown (ompA start codon in bold).

B. Hfq binding (blue), RNase E binding (grey), and oligoadenylation of the MicA binding site in the ompA mRNA. The MicA seed sequence is shaded blue.

C. Interaction strength (Gibbs free energy) of all RNA-RNA interactions recovered by RNase E-CLASH (solid line) and for randomly paired hybrid read halves (dashed line).

D. Interaction strength of sRNA-mRNA hybrid read halves (solid line) and randomly paired sRNA and mRNA read halves (dashed line).

E. Position of sRNA-mRNA interactions relative to the mRNA start codon.

F. Distribution of sRNA interactions recovered with each class of RNA.
Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E (2016)
Figure 4

Figure 4. sRNA-RNA interactions identify sRNA seed sequences.

A & B (top). Sequences interacting with ChiX (A) or RyhB (B) were analyzed for conserved motifs using MEME. Motifs that were enriched in the target RNAs and are shown above the heatmap with proportion of target RNAs carrying the motif (N) and the expected value for the motif (e-val). The complementary sequence motifs within ChiX or RyhB are shown below the logo. (Middle) Heatmaps showing the position of predicted basepairing for each interaction within ChiX or RyhB. Grey bars boxed below indicate the position of basepairing with experimentally verified mRNAs (known interactions). (Bottom) Hfq (blue) and RNase E (grey) binding sites within ChiX and RyhB. Hfq and RNase E-bound sequence, and non-genomically encoded oligo(A) tails within ChiX and RyhB are shown as line plots where the x-axis position correlates with heatmaps above.

C, D & E. Cumulative plots of oligoadenylation (C), Hfq (D) and RNase E (E) binding at predicted seed motifs.
Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E (2016)
Figure 5

Figure 5. Confirmation of high scoring sRNA-mRNA interactions and interactions with the EHEC-specific sRNA, Esr41.

A-H. Base pairing patterns for the sRNA-mRNA pairs identified by CLASH were calculated using IntaRNA software (Busch et al. 2008) and are shown (left) for frdA-RyhB (A), zapB-RyhB (B), hdeA-RyhB (C), hdeA-GadY (D), rssA-RyeB (E), chuA-Esr41 (F), cirA-Esr41 (G), and bfr-Esr41 (H). Point mutations and predicted sRNA seed sequences (blue shading) are indicated. (Histograms, right) Median fluorescence intensity (MFI) was assessed by FACS for mRNA-sfGFP fusions with compensatory base changes. In each case, introduction of a point mutation into an sRNA or mRNA construct (M1) is expected to reduce sRNA repression, which should be restored by combining complementary point mutants (last bar, M1-M1). A two-tailed t-test was used to calculate significance from biological triplicate cultures. *p<0.05.
Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E (2016)
Figure 6

Figure 6. The EHEC specific small RNA Esr41 regulates iron uptake, storage, and colicin resistance.

A-C. FACS analysis of constitutively expressed sfGFP fusions to chuA (A), cirA (B), or bfr (C) in the presence of Esr41 (orange), RyhB (blue), or a scrambled RNA control (red). The histogram shows GFP fluorescence for each sRNA-mRNA fusion. D. Esr41 confers resistance to Colicin 1A and 1B in the sensitive background, E. coli DH5alpha. Top agar lawns of DH5alpha expressing a control scrambled RNA (pJV300), Esr41 (pZE12::esr41), or RyhB (pZE12::ryhB) were spotted with colicins indicated (top). Zones of clearing indicate sensitivity to the tested colicin. E. Esr41 confers a competitive disadvantage on EHEC under iron-limiting conditions. Wild type EHEC and isogenic ∆esr41 strain (black), or EHEC ∆esr41 and the chromosomally repaired strain EHEC ∆esr41::esr41 were inoculated at equal densities and cultured in MEM-HEPES media for 3 days. The proportion of each strain was determined at each time point (days) and is expressed as a ratio relative to the starting inoculum where 1 is an equal fitness (see Appended Supplementary Methods).
Adaptive RSK-EphA2-GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer (2020)
Figure 1

Figure 1. Cisplatin treatment leads to EphA2 upregulation in ex vivo HGSC cultures.

A Confocal micrographs show CK7 (green) and vimentin (red) in patient-derived HGSC cells cultured in 3D collagen for 7 d. Scale bars: 50 μm.

B Confocal micrographs of EphA2 (red) in HGSC cells cultured in 3D collagen for 7 d and treated for 72 h with 10 μM cisplatin (5 μM for OCKI_p01). The intensity of EphA2 is comparable only between mock and treatment conditions for each patient. Scale bars: 20 μm.

C Chart illustrates EphA2 fold change after treatment. Mock is set to one. N = 3.

Data information: In (C) data are presented as mean fold change (SD). P-values provided in Appendix Table S10, Student's t-test.
Adaptive RSK-EphA2-GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer (2020)
Figure 2

Figure 2. OC cell lines and patient-derived cells undergo an EphA2 phosphorylation switch upon platinum treatment.

A, B EphA2 (total and phosphorylated at S897 or Y588) in OVCAR3, OVCAR4 and OVCAR8 after treatment with 0-20 μM cisplatin for 72 h was assessed by immunoblotting (A) and quantified for pS897/pY588 ratio (B). N = 4.

C EphA2 (total and phosphorylated) in corresponding untreated cells. The same β-actin detection for these samples is shown in Appendix Fig S1B.

D Cytotoxicity assay results after cell treatment with 0-20 μM cisplatin for 72 h. N = 6.

E Quantitative assessment of EphA2 (total and phosphorylated) and pS897/pY588 ratio in early passage patient-derived HGSC cultures treated with 0-20 μM cisplatin for 72 h (see immunoblots in Appendix Fig S2A). N = 6 patients, pooled.

F Corresponding EphA2 pS897/pY588 ratios for individual patient cells.

G, H EphA2 (total and phosphorylated) in OVCAR4 and OVCAR8 after treatment with 0-80 μM carboplatin for 72 h (G) along with pS897/pY588 quantification (H). N = 3.

Data information: In (B, D-E and H) data are presented as mean (SD). P-values provided in Appendix Table S10, Student's t-test.
Adaptive RSK-EphA2-GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer (2020)
Figure 3

Figure 3. Platinum treatment promotes ligand-independent oncogenic EphA2 phosphorylation in vivo.

A Bioluminescence images visualize mock and carboplatin treated OVCAR4 xenograft tumors (day 53; after 13 d i.p. treatment). Color scale unit: p/sec/cm2/sr.

B, C Charts illustrate the tumor growth (B) and ascites volumes at the end of the follow-up (C). Carboplatin treatment started on day 35. Bioluminescent unit: p/sec/cm2/sr. N = 5 mice/group.

D, E Representative micrographs of hematoxylin-eosin staining (D) and EphA2-pS897 immunofluorescence (E) in the xenografts tumors. Scale bars: 100 μm (D), 10 μm (E).

F, G Quantitative assessment of tumor EphA2 (F) and EphA2-pS897 (G). N = 5 mice/group.

H, I Confocal micrographs of tumor EphA2-pS897 (red) and cleaved caspase-3 (H; clCasp3, green) and corresponding clCasp3 quantification (I). Scale bars: 20 μm. N = 5 mice/group.

Data information: In (B-C, F-G and I) data are presented as mean (SD). P-values provided in Appendix Table S10, Mann Whitney U test.
Adaptive RSK-EphA2-GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer (2020)
Figure 4

Figure 4. Oncogenic EphA2 signaling switch is associated with platinum resistance and mediated by treatment-activated ERK1/2-RSK axis in OC cells.

A, B Immunoblot images show EphA2 (A; total, pS897 and pY588) and corresponding pS897/pY588 ratios (B) of TYK-nu and TYK-nu.R after treatment with 0-10 μM cisplatin for 72 h . N = 5.

C, D EphA2 (total and phosphorylated) in the cells treated (C) and untreated (D) with 0-80 μM carboplatin for 72 h.

E, F EphA2 (total and pS897) in TYK-nu after treatment with inhibitors against MEK (UO126, 10 μM), PKA (H89.2HCl, 10 μM), PI3K (LY294002, 10 μM) or its alpha subunit (BYL-719, 5 μM) and RSK (BI-D1870, 10 μM) in combination with 0-10 μM cisplatin for 72 h (E). Corresponding EphA2-pS897 quantification for BI-D1870 treatment (F) N = 5.

G EphA2 (total and pS897) in TYK-nu.R treated with 0-20 μM cisplatin without or with 10 μM BI-D1870 for 72 h.

H, I EphA2, RSK and ERK1/2 (total and phosphorylated) in TYK-nu (H) and OVCAR4 (I) treated with 0-30 μM carboplatin without or with 50 μM LJH685 for 72 h.

Data information: In (B and F) data are presented as mean (SD). P-values provided in Appendix Table S10, Student's t-test.
Adaptive RSK-EphA2-GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer (2020)
Figure 5

Figure 5. RSK-EphA2-pS897 inhibition enhances EphA2-pY588 and sensitizes OC cells to platinum chemotherapy.

A, B Charts show OC cell viability after 72 h treatment with 0-20 μM cisplatin and 10 μM BI-D1870 (A; N = 4) or with 50 μM LJH685 (B; N = 3) as indicated.

C RSK and EphA2 (total and phosphorylated) in OC cells treated with 10 µM cisplatin and 25 µM LJH685 as indicated for 72 h.

D Charts illustrate the corresponding quantified inhibition of EphA2-pS897 and increase of EphA2-pY588 shown in (C).

Data information: In (A-B) data are presented as mean (SD). P-values provided in Appendix Table S10, Student's t-test.
Adaptive RSK-EphA2-GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer (2020)
Figure 6

Figure 6. GPRC5A regulation defines OC cell responses to RSK1/2-EphA2-pS897 inhibition.

A, B Immunoblot images (A) and quantification (B) show GPRC5A 46/41 kDa ratios in OC cells treated with 0-10 µM cisplatin and with 25 µM LJH685 alone or as a combination for 72 h. The ratio in mock cells was set to 1. N = 4.

C Confocal micrographs of EphA2 (red) and GPRC5A (green) in TYK-nu and TYK-nu.R treated with 5 µM cisplatin and 10 μM LJH685 as indicated for 72 h. Arrows point co-localization of the receptors. Scale bars: 20 μm.

D, E EphA2 (total and phosphorylated), RSK1/2 and GPRC5A in TYK-nu (D) and these proteins coupled with PARP and PCNA in TYK-nu.R (E) were assessed by immunoblotting after RSK1/2 knockdown for 2 d and following treatment with 0-5 μM cisplatin for 72 h. Asterisks indicate unspecific bands.

F Chart illustrates the viability of the RSK1/2 depleted TYK-nu.R after cisplatin treatment. N = 3.

Data information: In (B and F) data are presented as mean (SD). P-values provided in Appendix Table S10, Student's t-test.
Adaptive RSK-EphA2-GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer (2020)
Figure 7

Figure 7. RSKi combinatorial treatment induces apoptosis in 3D co-culture and in vivo.

A Confocal micrographs show cleaved caspase-3 (clCasp3, green), mRFP (orange) and phalloidin (F-actin, red; only shown for CAF) in 3D OVCAR8-RFP and OCKI_p22 CAF mono- and co-cultures treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 100 μm.

B Quantitative assessment of clCasp3+ cells in the 3D cultures. N = 4.

C Confocal micrographs show cytokeratin 7 (CK7, green) and phalloidin (F-actin, red) in 3D patient OCKI_p13 mono- and co-culture with OCKI_p22 CAF treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 50 μm.

D Quantitative assessment of clCasp3+ OCKI_p13 cells in 3D co-cultures of patient-derived cells. N = 4.

E-H Immunohistochemistry images and quantifications of TUNEL (E-F) and Ki67 (G-H) in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm.

Data information: In (B and D, F and H) data are presented as mean (SD). P-values provided in Appendix Table S10, Student's t-test (B, D) and Mann Whitney U test (F, H).