Journal: Molecular Systems Biology

Article Title: AI-guided pipeline for protein–protein interaction drug discovery identifies a SARS-CoV-2 inhibitor

doi: 10.1038/s44320-024-00019-8

Figure Lengend Snippet: ( A ) Receiver characteristic analysis comparing sensitivity and specificity between the five AFM-predicted structural models for PAE, ΔG and iA of the hsPRS-AF and hsRRS-AF. ( B ) Bar plots showing the fraction of hsPRS-AF and hsRRS-AF interactions with structures deposited in PDB that scored above classifier probabilities of 50%, 75% and 95% by AlphaFold-Multimer (i) by LuTHy (ii) or the mean recovery of N2H (MN2H, VN2H, YN2H), GPCA, KISS, MAPPIT and NanoBiT (iii). Data for the SIMPL assay was excluded for this analysis due to the different composition of the reference sets. LuTHy experiments from this study were repeated two times with n = 2, biological replicates, each containing n = 3 technical replicates; AFM was used to predict n = 5 structural models; all other from Choi et al (Choi et al, ). Bars and error bars in this figure represent mean values and standard error of the proportion, respectively.

Article Snippet: Entry clones were shuttled into LuTHy (Addgene #113446, #113447, #113448, #113449) and N2H (Addgene #125547, #125548, #125549, #125559) destination vectors using the Gateway Cloning Technology.

Techniques:

Journal: Molecular Systems Biology

Article Title: AI-guided pipeline for protein–protein interaction drug discovery identifies a SARS-CoV-2 inhibitor

doi: 10.1038/s44320-024-00019-8

Figure Lengend Snippet: Reagents and tools.

Article Snippet: Entry clones were shuttled into LuTHy (Addgene #113446, #113447, #113448, #113449) and N2H (Addgene #125547, #125548, #125549, #125559) destination vectors using the Gateway Cloning Technology.

Techniques: Recombinant, Control, Sequencing, Cell Culture, Binding Assay, Protease Inhibitor, Software

Journal: The Journal of Experimental Medicine

Article Title: A TBK1 variant causes autophagolysosomal and motoneuron pathology without neuroinflammation in mice

doi: 10.1084/jem.20221190

Figure Lengend Snippet: Characterization of the TBK1 E696K variant. (A) Scheme of LuTHy-BRET assay to investigate binding of wt TBK1 and TBK1 E696K to optineurin (OPTN) or the TRAF family member–associated NF-κB activator (TANK) in live HEK293 cells. (B and C) Binding of wt TBK1- and TBK1 E696K -mCitrine-Protein A (-mCit-PA) to NanoLuc (NL)-tagged optineurin (B) or TANK (C) in LuTHy-BRET donor saturation assays. (D) Quantification of cBRET signals from TBK1 binding assays. PA-mCit-NL tandem construct shown as positive and cotransfection of single NL- and PA-mCit-tags as negative BRET controls. Relative BRET ratios were obtained by normalizing the BRET signal of optineurin or TANK with each TBK1 mutant to the interaction signals with wt TBK1, respectively. Mean ± SEM of n = 4 technical replicates per condition from three independent experiments; one-way ANOVA with Sidak’s post hoc test; **P < 0.01. (E) Comparison of wt TBK1- and TBK1 E696K -mCit-PA fusion protein expression as measured from fluorescence intensities. Mean ± SEM of n = 7 technical replicates per condition from three independent experiments; Student’s t test. (F) Western blot showing expression of the TBK1 mutants in HEK293 cells after transfection. Source data are available for this figure: .

Article Snippet: LuTHy control vectors expressing only NL (#113442; Addgene) or PA-mCit ( #113443; Addgene) were used for the calculation of corrected scores, the PA-mCit-NL tandem construct (#113444; Addgene) as positive, and NL cotransfected with PA-mCit-only as negative controls.

Techniques: Variant Assay, Bioluminescence Resonance Energy Transfer, Binding Assay, Construct, Cotransfection, Mutagenesis, Comparison, Expressing, Fluorescence, Western Blot, Transfection

Journal: bioRxiv

Article Title: Expanding the Huntington’s disease research toolbox; validated subdomain protein constructs for biochemical and structural investigation of huntingtin

doi: 10.1101/2022.11.21.516512

Figure Lengend Snippet: A, Graphical illustration of the LuTHy-BRET assay performed. HTT FL or HTT CTD were expressed as mCitrine-Protein A (mCit-PA)- and HAP40 as NanoLuc luciferase (NL)-tagged fusion proteins in HEK293 cells. After expression for 48 h and addition of luciferase substrate, BRET was quantified from live cells. B, BRET ratios between HAP40-NL and HTT FL-mCit-PA and HTT CTD-mCit-PA. As a control, NL only was co-transfected with the HTT acceptor constructs, respectively, as well as PA-mCit only with the HAP40-NL donor construct. HAP40-NL co-expressed with HTT FL-mCit-PA and HTT CTD-mCit-PA showed significantly increased BRET ratios compared to controls, respectively. Bars represent means ± SEM from two independent experiments performed in triplicate. One-way ANOVA with Tukey’s multiple comparisons test, **p < 0.002, *** p < 0.001.

Article Snippet: To generate LuTHy expression plasmids, HAP40 was cloned into a LuTHy donor vector (Addgene plasmid #113447) and the HTT constructs into a LuTHy acceptor vector (Addgene plasmid #113449) by LR Clonase reactions (Gateway Cloning System, Invitrogen).

Techniques: Bioluminescence Resonance Energy Transfer, Luciferase, Expressing, Transfection, Construct

Journal: Molecular Systems Biology

Article Title: AI-guided pipeline for protein–protein interaction drug discovery identifies a SARS-CoV-2 inhibitor

doi: 10.1038/s44320-024-00019-8

Figure Lengend Snippet: Reagents and tools.

Article Snippet: pcDNA3.1 LuTHy destination and control vectors , Addgene (Trepte et al, ) , 113442–113449.

Techniques: Recombinant, Control, Sequencing, Cell Culture, Binding Assay, Protease Inhibitor, Software

Journal: bioRxiv

Article Title: A quantitative mapping approach to identify direct interactions within complexomes

doi: 10.1101/2021.08.25.457734

Figure Lengend Snippet: ( A ) Schematic for the definition of direct interactions and indirect associations within complexes using structural biology data. ( B-D ) Distribution of the quantitative scores and comparison between direct interactions and indirect associations. Direct interactions produce significantly higher LuTHy-BRET ( B ), LuTHy-LuC ( C ), and mN2H ( D ) scores compared to indirect associations (Welch’s two-tailed t-test). ( E ) Scatter plot of cBRET and cLuC ratios showing direct interactions (diamonds) and indirect associations (circles) detected by LuTHy-BRET or LuTHy-LuC. Protein pairs were clustered by supervised expectation-maximization clustering as direct (red) or not-direct (blue) interactions using hsPRS-PDB and hsRRS-v2 as training sets. ( F ) Recovery and classification of structurally defined direct interactions as true direct interactions or as indirect associations by BioPlex (AP-MS), LuTHy and mN2H. ( G ) Pearson correlation matrix for direct interactions comparing cBRET, cLuC and mN2H ratios to the interaction interface areas (Å 2 ), or to the total complex surface areas (Å 2 ). ( H ) 3D structure of the DR1-DRAP1 interaction (PDB: 1JFI). Full-length DR1 but only the N-terminal region of DRAP1 (1-77, amino acids 78-205 lacking) are reported in the 3D structure. The cBRET ratios were used to interpolate distances between the C-terminus of DRAP1 and the N-terminus (60.7 Å), or C-terminus (73.8 Å) of DR1 based on the cBRET-distance standard curve. ( I ) cBRET ratios for the DR1-DRAP1 PPI are plotted against the molecular distances obtained from the 3D structure. Tagging configurations are colored by N-N (grey), C-C (green), N-C (purple) or C-N (yellow). Each data point on the graph is labeled (framed text) according to the tested tagging configuration: the protein indicated first is tagged with NanoLuc (NL) luciferase, while the second protein is tagged with PA-mCitrine (PA-mCit) (e.g. DR1+DRAP1 (N-N, grey) corresponds to NL-DR1/PA-mCit-DRAP1). Tagging configurations where tags are fused to the structurally unresolved termini in the current 3D structure for one of the two proteins are outlined in red (e.g. DR1+DRAP1 (N-C, purple) corresponds to NL-DR1/DRAP1-mCit-PA). Interaction values outlined in purple represent cBRET ratios against interpolated molecular distances. Differences between structurally determined and interpolated distances are indicated by dotted arrows. ( J ) cBRET ratios plotted against the structurally determined molecular distances for the 44 protein pairs used as references (blue). The 30 PPIs with tags fused to protein termini not currently resolved in the structures are plotted against interpolated distances between full-length proteins (purple). For each tested pair, the purple horizontal error bar corresponds to the 95% confidence interval of the interpolated molecular distances. Sigmoidal fits were performed on the 44 tested pairs outlined in black, together with the 30 pairs outlined in purple (R = −0.70).

Article Snippet: For the LuTHy assay, additional control plasmids (PA-NL, Addgene #113445; PA-mCit-NL, Addgene #113444; PA-mCit, Addgene #113443; NL, Addgene #113442) were used, as previously described .

Techniques: Comparison, Two Tailed Test, Protein-Protein interactions, Labeling, Luciferase

Journal: bioRxiv

Article Title: A quantitative mapping approach to identify direct interactions within complexomes

doi: 10.1101/2021.08.25.457734

Figure Lengend Snippet: Protein sequence coverage and molecular distances between subunits of the studied complexes used to generate the cBRET-distance standard curve. ( A ) Relative distribution of structure-based distances for direct interactions within the LAMTOR, BRISC and MIS12 complexes, as well as in the hsPRS-PDB PPIs. ( B ) Proportion of directly interacting proteins reported in the 3D structures that contain, on average, more (white) or less (green) than 75% of the full-length protein sequences. ( C ) Correlation between the cBRET ratios and the molecular distances for the 44 protein pairs where at least six tagging configurations out of the eight tested are scored positive in LuTHy-BRET, and for which the respective tagged protein termini are structurally resolved. Goodness of the sigmoidal regression fit is indicated on the graph (R = −0.58). ( D ) 3D structure of the DR1-DRAP1 interaction where molecular distances between the last amino acids of the structurally solved proteins are indicated in red. The dotted blue lines indicate the protein fractions that are not structurally resolved (DRAP1 C-terminus). Unknown molecular distances between the missing termini in the 3D structure are indicated by dotted purple lines. ( E ) cBRET ratios for the DR1-DRAP1 interaction are plotted against the structure-based molecular distances. Tagging configurations are colored by N-N (grey), C-C (green), N-C (purple) or C-N (yellow). Each data point on the graph is labeled (framed text) according to the tested tagging configuration: the protein indicated first is tagged with NanoLuc (NL) luciferase, while the second protein is tagged with PA-mCitrine (PA-mCit) (e.g. DR1+DRAP1 (N-N, grey) corresponds to NL-DR1/PA-mCit-DRAP1). Tagging configurations where tags are fused to the structurally unresolved termini in the current 3D structure for one of the two proteins are outlined in red (e.g. DR1+DRAP1 (N-C, purple) corresponds to NL-DR1/DRAP1-mCit-PA). ( F ) Structure-based and predicted distances for the 30 PPIs with tags fused to protein termini not currently resolved in the structures (box and whisker visualizing the median, lower and upper hinges showing the 25 th and 75 th percentiles, lower and upper whiskers extending from min to max; all data points are shown). Statistical significance was calculated by a two-tailed, paired t-test, n=30.

Article Snippet: For the LuTHy assay, additional control plasmids (PA-NL, Addgene #113445; PA-mCit-NL, Addgene #113444; PA-mCit, Addgene #113443; NL, Addgene #113442) were used, as previously described .

Techniques: Sequencing, Labeling, Luciferase, Whisker Assay, Two Tailed Test

Journal: bioRxiv

Article Title: A quantitative mapping approach to identify direct interactions within complexomes

doi: 10.1101/2021.08.25.457734

Figure Lengend Snippet: Subset of interactions where at least six out of the eight tested tagging configurations scored positive in the LuTHy-BRET assay. The following 3D structures are represented: 1G5J (BAD-BCL2L1), 1BUH (CDK2-CKS1B), 1JFI (DR1-DRAP1), 1F3U (GTF2F1-GTF2F2), 1A02 (JUNB-BATF), 6EHR (LAMTOR1-LAMTOR2 and LAMTOR3-LAMTOR2), 4M7D (LSM3-LSM2), 5LSJ (MIS12-PMF1), and 6EHR (RRAGA-RRAGC). On each structure, the protein termini are indicated by N or C. Protein sequence coverages used in the structures are also indicated (%). Protein regions that are missing in the structures are indicated by dotted lines. For all interactions, cBRET ratios are plotted against the molecular distances measured in the corresponding 3D structures or interpolated using the cBRET standard curve. The quantitative scores outlined in black correspond to tagging configurations where the tagged protein termini are resolved in the structures. These scores were used as reference data points for distance interpolations. Interaction scores and structure-based molecular distances for tagging configurations where the tagged protein termini are absent from the 3D structure are outlined in red. Interaction scores outlined in purple correspond to cBRET ratios against interpolated molecular distances. Changes in structurally measured distances versus the interpolated distances are indicated by dotted arrows. The purple horizontal error bars correspond to the 95% confidence intervals of the interpolated molecular distances. Tagging configurations are color-coded: N-N (grey), C-C (green), N-C (purple), and C-N (yellow). Each data point on the graph is labeled (blank frame) according to the associated tagging configuration tested: the protein indicated first is tagged with NanoLuc (NL) luciferase, while the second protein is tagged with PA-mCitrine (PA-mCit) (e.g. DR1-DRAP1 (N-N, grey) corresponds to NL-DR1/PA-mCit-DRAP1). Tagging configurations where tags are fused to the missing termini in the current 3D structure for one of the two proteins are outlined in red (e.g. DR1-DRAP1 (N-C, purple) corresponds to NL-DR1/DRAP1-mCit-PA). The size of the dots indicates the average percentage of protein sequence coverage in the corresponding 3D structure of the PPI.

Article Snippet: For the LuTHy assay, additional control plasmids (PA-NL, Addgene #113445; PA-mCit-NL, Addgene #113444; PA-mCit, Addgene #113443; NL, Addgene #113442) were used, as previously described .

Techniques: Bioluminescence Resonance Energy Transfer, Sequencing, Labeling, Luciferase