Figures and data
Overview of Orf9b homodimer crystallization system and fragment screening.
A. Model for the Tom70-Orf9b equilibrium. Orf9b homodimer dissociates into Orf9b monomers which undergo a conformational change from β-sheet to ɑ-helix. ɑ-helical Orf9b binds to Tom70. B. Overview of Orf9b homodimer with modeled fragment hits. Orf9b homodimer surface is modeled with fragments as sticks including the number of modeled hits per binding site. Hydrogen bonds are shown as black dashes with a 4Å cutoff. C. Overview of binding site 1 showing residue side chains. Representative binding poses of modeled fragments bound at site 1. PanDDA event maps are shown contoured to 2σ. Hydrogen bonds are shown as black dashes with a 3.5Å cutoff. D. Representative binding poses of fragments identified at sites 3 and 4 corresponding to the homodimer central channel, the lipid molecule colored in green is modeled to provide orientation. PanDDA event maps are shown contoured to 3σ. Hydrogen bonds are shown as black dashes with a 3.5Å cutoff. E. Overview of binding site 2 showing representative fragments with PanDDA event maps contoured at 2σ. Fragment in structure PDB 13TT (ii) is shown with two conformations.
Overview of Orf9b homodimer analog binding and binding behaviors.
A. Structurally resolved fragment hit and analog compounds bound at site 1 with FTMap chemical probes superimposed. PDB codes for deposited structures are displayed in the top left corner of each panel. B. (Top) Kinetic traces of Orf9b homodimer binding to Tom70 in the presence of increasing x436 analog concentration. (Bottom left) Diagram of SPR experiment with immobilized Orf9b homodimer and x436 analog compound to measure binding at external dimer interface. (Bottom middle) Surface plasmon resonance sensorgram showing x436 analog compound binding to the Orf9b homodimer immobilized on the surface. (Bottom Right) Modeling SPR sensorgram equilibrium response with a non-linear regression to determine the affinity for the Orf9b homodimer of 17uM. C. (Top) Chai1 predictions of Orf9b homodimer co-folded with one copy of lipidated analog and ipTM scores in the Orf9b homodimer central channel. (Bottom) Chai1 predictions of Orf9b homodimer co-folded with two copies of the lipidated analog and ipTM scores. The original fragment and modeled lipid are highlighted in green with the Chai1 predicted analogs in cyan. D. Kinetic model overlay to FP competition kinetic assay using Orf9b homodimer incubated with lipidated analog as the competitor. Structures of lipidated analogs that exhibit slow homodimer dissociation.
High-throughput screen identifies Tom70-binding small molecules that block Orf9b binding.
A. Diagram for generating fluorescent peptides derived from Orf9b for high throughput screening against Tom70. B. Overview of high throughput screen and triage process. C. Dose response curves for compounds identified by high throughput screen. D. Chai-1 prediction of Tom70 co-folded with HTS hits (green and blue). Orf9b (magenta) is superimposed with compounds illustrating potential mechanisms for blocking Orf9b binding to Tom70. E. Diagram of surface plasmon resonance experiments to measure binding of Tom70 to immobilized Orf9b peptide in the presence or absence of competitive binders derived from the Orf9b peptide. F. SPR sensorgrams for Tom70 binding to Orf9b peptide immobilized surface in the presence or absence of competitive binders.
Summary of Tom70-binding analog compounds.
A. Dose response of select HTS analog compounds in FP format. B. Table of purchased analogs showing the relationship between changes to the benzene group and affinity for Tom70. Compounds that had no measurable effect on FP were reported to have Ki >200uM. C. Chai-1 predictions of Tom70 co-folded with top binding analogs with ipTM scores. Original HTS hits co-folded with Tom70 are shown in blue and green and analog compounds are shown in cyan.
Summary of DMSO and fragment soaked data sets.
A. Orf9b homodimer crystal system unit cell parameters. All datasets collected showed less than 1% variation in unit cell dimensions. B. Distribution of resolution ranges collected for DMSO soaked crystals used for PanDDA background maps. C. Distribution of resolution ranges for fragment soaked datasets collected for conducting PanDDA analysis.
Comparison of fragment binding poses in sites 1 and 2.
A. Comparison of fragment binding sites 1 and 2. Binding site 2 is partially occupied by a DMSO molecule and has fragments that are capable of forming favorable hydrogen bonds with the neighboring symmetry mate. B. Superimposition of all modeled fragments from sites 1 onto site 2. Fragments at site 2 (cyan) bind in a region and orientation in the binding site that is distinct to site 1 fragments (green) C. Comparison of neighboring symmetry mates at fragment binding sites 3 and 4. Binding site 3 fragments are approximately 22A away from the nearest symmetry mate whereas site 4 fragments are in close proximity to a symmetry mate near the central channel entrance. D. Observed conformational change of R47 on chain B upon fragment binding. Differences in fragment binding poses observed in sites 1 and 2 with neighboring symmetry mate. E. Electron density modeled as DMSO occupies part of binding site 2 reducing pocket size for fragment binding.
Overview of FTMap predicted fragment hot-spots.
A. Distribution of FTMap probes bound at site 1 on the Orf9b homodimer. B. Distribution of FTMap probes bound at site 2 on the Orf9b homodimer. C. Distribution of FTMap probes bound to the central channel of the Orf9b homodimer.
Lipidated analogs do not act on monomeric Orf9b or Tom70 directly.
A. Lipidated Orf9b analogs do not compete with the fluorescent probe for binding to Tom70 in kinetic assay format. Control peptide exhibits a sharp decrease in FP signal over time as a positive control. B. Lipidated Orf9b analogs do not interfere with Orf9b peptides from binding to Tom70 in kinetic assay format.
Design of fluorescent Orf9b peptides and HTS screen.
A. Design of fluorescent peptides derived from WT Orf9b. The structurally resolved residues of Orf9b bound to Tom70 (44-70) were used for generating C-terminally appended fluorescein peptides. A further 10 amino acids are truncated from the C-terminus of the Orf9b-FITC construct to generate the Orf9bΔ10aa-FITC fluorescent peptide used in the high throughput screen. B. Titration of Tom70 against a fixed concentration of Orf9bΔ10aa-FITC was performed to identify the Kd. A non-linear regression single binding site model was used to determine the Kd of 3.4uM C. Overview of a high-throughput screen set up. HTS was performed in 384 well plates with all wells containing the Tom70:Orf9bΔ10aa-FITC complex. Columns 1-2 contain only DMSO added and columns 23-24 contain 10uM of the Orf9b peptide as a positive control. Z’ values were calculated from columns 1-2 and 23-24.
Structure predictions for Tom70-small molecule binding sites.
A. FTMap probe clusters within the Tom70 C-terminal binding domain. Clusters are grouped and number 1-3. B. Superimposition of Chai-1 predicted binding locations for HTS Hits 1 and 2 with FTMap probe clusters. C. Superimposition of Chai-1 predicted binding locations for HTS Hits 1 and 2 with FTMap probe clusters and Orf9b from the Cyro-EM structure of the Orf9b:Tom70 complex (PDB 7DHG).
Dissociation Constants and Competition Binding Curves of Tom70 Analog Compounds Competition binding curves of all tested Tom70 analog compounds in FP format.
Error bars are for duplicate measurements with fitted curves shown using a one site fit for calculating Ki values. Structures of the compounds tested are shown in the bottom left.
