Journal: iScience

Article Title: Small-molecule activators of the Staphylococcus aureus ClpC/ClpP AAA+ protease

doi: 10.1016/j.isci.2026.117738

Figure Lengend Snippet:

Article Snippet: After cell lysis using a French press, the cell debris was removed by centrifugation at 18,000 g for 60 min at 4°C, and the cleared lysate was subsequently transferred into a plastic column containing pre-equilibrated 0.8–1 g Protino Ni-IDA resin (Macherey-Nagel) at 4°C.

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Cloning, Mutagenesis, Sequencing, Software, Analysis, Microscopy

Journal: bioRxiv

Article Title: Identification and targeting of a novel drug pocket in the tumour angiogenesis regulator RhoJ

doi: 10.64898/2026.10.14.758331

Figure Lengend Snippet: (A) Multiple-sequence alignment (MSA) of human Cdc42 subfamily GTPases (Cdc42, RhoQ, and RhoJ) performed with T-Coffee. Residues are shaded by conservation (yellow). Structural and regulatory motifs are indicated: G-boxes G1–G5 (green), Switch Region I (dark blue), Switch Region II (blue), Rho-insert region (tan), hypervariable C-terminus (hot pink), polybasic region (light pink), and CaaX box (grey). The RhoJ-specific N-terminal extension is highlighted (cyan). Cysteine residues are colored by predicted post-translational modification: S-palmitoylation (dark cyan), S-farnesylation (rose), and S-geranylgeranylation (olive). Residues comprising the previously predicted allosteric binding pocket are colored in dark red. (B) Representative structures of activated GTPases complexed with the PBD PAK3 . (Left) Lowest-RMSDC α replica of the RhoJ-Mg 2+ -GppCp:PBD PAK3 complex from all-atom MD simulations at 500 ns. (Right) NMR structure of the Cdc42:PBD PAK3 complex (PDB: 1EES) for comparison. GTPases are shown in surface representation (core: white; RhoJ N-terminus Met1-Lys20: cyan; PBD PAK3 interface: medium slate blue). PBD PAK3 is shown as a red ribbon/stick model. (C) MD stability and flexibility of the activated RhoJ:PBD PAK3 complex. Left, mean RMSDC α over 0-500 ns; right, per-residue RMSFC α over the equilibrated window (150-500 ns). Data are mean ± SEM ( n = 3 independent simulations).

Article Snippet: 2.4 ng of purified recombinant constitutively active Cdc42 protein (CDCA, Cytoskeleton Inc.) in lysis buffer (GL35, Cytoskeleton Inc.) was treated with 5% (v/v) DMSO or terconazole for 15 min at 4°C.

Techniques: Sequencing, Modification, Binding Assay, Comparison, Stability, Residue

Journal: bioRxiv

Article Title: Identification and targeting of a novel drug pocket in the tumour angiogenesis regulator RhoJ

doi: 10.64898/2026.10.14.758331

Figure Lengend Snippet: (A) Close-up 3D view of interaction hotspots within the RhoJ:PBD PAK3 N-terminal pocket from the 500 ns MD simulation frame. PAK3 residues Leu72, Pro73, and Ser74 are highlighted in maroon; the RhoJ N-terminus is cyan, and interacting residues are medium slate blue. Cyan dashed lines indicate hydrogen bonds. (B) 2D interaction map of N-terminal pocket hotspots. The black dashed line represents the protein-protein interface. Hydrogen bonds are shown as green dotted lines (lengths in Å); hydrophobic interactions are indicated by red radial sectors. (C) Comparative computational alanine scanning (CAS) of PBD PAK3 binding to RhoJ and Cdc42 with quantification of displacement of PBD PAK3 . The red line represents , calculated as the difference in average normalized per-residue binding energy change between the RhoJ:PBD PAK3 MD models and the Cdc42:PBD PAK3 NMR structure (PDB: 1EES) . Blue points indicate the per-residue C α displacement of PBD PAK3 between the aligned RhoJ and Cdc42 complexes. CAS was performed using BudeAlaScan (BAlaS). Δ(Δ G R ℎ oJ / Cdc 42: PBD PAK 3 ) X → Ala was calculated using the following equation: . The brown dashed box highlights the and Cα displacements for PAK3 Leu72–Ser74. (D) CAS of RhoJ interface residues across triplicate 500 ns-long MD simulations. (E) Quantification of N-terminal contribution to RhoJ:PBD PAK3 interface. The N-terminal contribution to the RhoJ:PBD PAK3 interface was estimated by comparing full-length, 500ns MD-derived RhoJ:PBD PAK3 models against the corresponding models with a direct in silico truncation of the RhoJ N-terminal residues (Met1-Lys20). The N-terminus accounts for a buried surface area of 285.6 Å 2 interface calculated by PDBePISA, representing ∼17.0% of total RhoJ:PBD PAK3 interface. (F, G) Structural and energetic impacts of RhoJ N-terminus on the RhoJ:PBD PAK3 interface within a physiological membrane context. Buried surface area (F) and predicted solvation free-energy gain upon interface formation (Δ i G) (G) were quantified across triplicate 500 ns on-membrane MD simulations using PDBePISA. N-terminal S-palmitoylation significantly reduced the buried interface area by an average of 213.4 Ų (∼14.0% of the total RhoJ:PBD PAK3 interface area), and significantly decreased the magnitude of Δ i G by ∼17.7%, indicating less favourable interface formation. Data are presented as mean ± SEM (n = 3 independent simulations). Statistical significance was determined by a two-tailed Welch’s unpaired t-test (*p < 0.05).

Article Snippet: 2.4 ng of purified recombinant constitutively active Cdc42 protein (CDCA, Cytoskeleton Inc.) in lysis buffer (GL35, Cytoskeleton Inc.) was treated with 5% (v/v) DMSO or terconazole for 15 min at 4°C.

Techniques: Binding Assay, Residue, Derivative Assay, In Silico, Membrane, Two Tailed Test

Journal: bioRxiv

Article Title: Identification and targeting of a novel drug pocket in the tumour angiogenesis regulator RhoJ

doi: 10.64898/2026.10.14.758331

Figure Lengend Snippet: For all panels: Left, surface representation of the lowest-RMSDC α 500ns-long MD model of activated RhoJ (white; N-terminus Met1–Lys20, cyan; PBD-contact residues, medium slate blue). Right, reference structure of activated Cdc42 (ivory; PBD-contact residues, dark slate blue). Bottom, effector positional shifts quantified by RMSDC ɑ when superimposing of RhoJ and Cdc42 complexes. (A) PBD PAK3 (red in RhoJ complex; dark red in Cdc42 NMR structure, PDB: 1EES). (B) PBD PAK1 (orange in RhoJ complex; dark orange in Cdc42 NMR structure, PDB: 1E0A). (C) PBD N-WASP (olive in RhoJ complex; dark olive green in Cdc42 MD-model). (D) PBD BORG3 (gray in RhoJ complex; slate gray in Cdc42 MD-model). All models represent the activated (Mg 2+ -GppCp or GppNHp bound) state.

Article Snippet: 2.4 ng of purified recombinant constitutively active Cdc42 protein (CDCA, Cytoskeleton Inc.) in lysis buffer (GL35, Cytoskeleton Inc.) was treated with 5% (v/v) DMSO or terconazole for 15 min at 4°C.

Techniques:

Journal: bioRxiv

Article Title: Identification and targeting of a novel drug pocket in the tumour angiogenesis regulator RhoJ

doi: 10.64898/2026.10.14.758331

Figure Lengend Snippet: Mean PDBePISA-predicted interface binding energies (Δ i G) for Cdc42 and RhoJ complexes with the indicated effector-binding domains (PBDs). Each value represents the mean Δ i G calculated from the 500-ns endpoint snapshot of each of three independent MD simulations per complex.

Article Snippet: 2.4 ng of purified recombinant constitutively active Cdc42 protein (CDCA, Cytoskeleton Inc.) in lysis buffer (GL35, Cytoskeleton Inc.) was treated with 5% (v/v) DMSO or terconazole for 15 min at 4°C.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Identification and targeting of a novel drug pocket in the tumour angiogenesis regulator RhoJ

doi: 10.64898/2026.10.14.758331

Figure Lengend Snippet: (A) eBRET 2 -based screening of top virtual hits targeting RhoJ N-t pocket. HEK293T cells expressing the RhoJ biosensor (RhoJ Q79L –RLuc8–Ct : GFP 2 –PBD PAK3 , donor:acceptor ratio = 1:10) were treated with candidates (200 µM), DMSO (1% v/v; vehicle control), or ML141 (20 µM; inhibition control) for 30 min. Statistics: one-way ANOVA with Bonferroni post-hoc test (ns, not significant; *p < 0.05, ****p < 0.0001). n = 3 independent experiments. (B) Dose-response characterization of terconazole. Corrected BRET ratios were fitted to a 4-parameter logistic regression, , yielding a = BRET max = 518 ± 12.9 mBU, b = −3.06 ± 0.301, c = IC 50 = 101 ± 6.80 μM, and d = 72.1 ± 2.27 mBU. Dotted lines indicate 95% confidence intervals of the fitted curve. n = 3 independent experiments. (C) Predicted binding mode of terconazole within the RhoJ N-terminal pocket. Terconazole (coral sticks) was docked into a representative MD snapshot (450 ns, with a BUDE-predicted ΔG closest to the mean across the 5 dockings). RhoJ is shown in surface representation (medium slate blue; N-terminus, cyan; Rho-insert region, tan; C-terminus, hot pink; previously reported allosteric pocket residues, blue). Mg 2+ and GppCp are shown as lime sphere and tan sticks, respectively. Inset: Detailed view of the binding site with labelled contacting residues. (D) 2D interaction fingerprint of the terconazole docking pose from (C), illustrating hydrophobic contacts (red radial sectors) with surrounding residues. (E) Selectivity assessment of terconazole against Cdc42. Purified constitutively active Cdc42 was incubated with vehicle [5% (v/v) DMSO] or terconazole for 15 min. Binding of Cdc42 to immobilised PBD PAK1 was quantified by G-LISA assay. Terconazole produced no significant alteration in Cdc42 binding to PBD PAK1 compared to vehicle control. n = 4 independent experiments. Statistical significance was assessed using a Geisser-Greenhouse-corrected one-way repeated-measures ANOVA followed by Dunnett’s post-hoc test against the vehicle control (ns, not significant). Data are mean ± SEM.

Article Snippet: 2.4 ng of purified recombinant constitutively active Cdc42 protein (CDCA, Cytoskeleton Inc.) in lysis buffer (GL35, Cytoskeleton Inc.) was treated with 5% (v/v) DMSO or terconazole for 15 min at 4°C.

Techniques: Screening, Expressing, Control, Inhibition, Binding Assay, Docking Assay, Purification, Incubation, Produced, Bioluminescence Resonance Energy Transfer