Biacore 4000

The equilibrium-binding constant (KD) of FeTPPS or PIX and HMGB1 was determined by Open SPR conducted using a BIAcore4000 instrument (BIAcore). All the steps were performed according to the previously described protocol^{14 (link)}.

Surface plasmon resonance binding assays were performed using a BIAcore 4000 instrument, in SPR buffer as follows: 10mM HEPES, 150mM NaCl, 3.4mM EDTA, 0.005% Tween‐20, pH 7.4. Monoclonal antibodies were immobilised as the ligand on a CM5 sensor chip surface by amine coupling at densities ranging from 1,516 to 5,574 RU. Sensorgrams were double referenced and initially fitted to a Langmuir specific one‐site binding model and then a heterogeneous ligand‐binding model, if appropriate, to derive on and off rates and the dissociation constants (K_D). Competitive assays were performed by pre‐incubating the analyte with the varying concentrations (78 to 1250 ng ml⁻¹) of the competing antibody with either CyRPA or PfRipr at a constant 8nM prior to flowing over the ligand immobilised surface. Kinetic parameters and competition experiments were derived from three experiments performed on independent days.

Wild‐type ITK was immobilized onto an NTA sensor chip using the capture coupling method that results in the capture in a nonrandom orientation by the His₆‐tag prior to amine coupling to produce functional and stable surfaces 31. The surface of the sensor chip was activated with a mixture of 0.1 mol·L⁻¹N‐hydroxysuccinimide and 0.4 mol·L⁻¹N‐ethyl‐N’‐(dimethylaminopropyl) carbodiimide for 5 min at 10 °C. WT ITK (10 μg·mL⁻¹) in running buffer (25 mm HEPES, pH 7.5, 25 mm KCl, 10 mm MgCl₂, 1 mm DTT, 0.005% Tween 20) was injected for 10 min. Typical immobilization levels ranged from 2900 to 4900 resonance units.
The compounds were diluted directly into the running buffer containing 5% DMSO. Serially diluted compounds were injected in a separate cycle for 60‐s association followed by 60‐s dissociation at a flow rate of 30 μL·min⁻¹. Competition experiments were carried out in the presence of ADP at a 1000 μm concentration diluted in the running buffer.
All data analysis was performed using Biacore T200 or Biacore 4000 evaluation software. Sensorgram data were solvent‐corrected and double‐referenced. For kinetic analysis, data were fit to 1 : 1 interaction model. For steady‐state analysis, responses at equilibrium were plotted against the compound concentration and fit to a 1 : 1 Langmuir binding isotherm.

Surface plasmon resonance experiments were performed using a Biacore 4000 instrument at a constant temperature of 25 °C. Sensor chips, buffer stock solutions and immobilisation reagents were purchased from GE Healthcare.

Molecular docking, a computational approach to calculate the binding energy between proteins and ligands [23 (link)], were used for screening the target of PHZ-OH. Given that OMV mediates cytosolic delivery of LPS by clathrin-dependent endocytosis [12 (link)], the crystal structures of potential targets, including LPS-related receptors or endocytosis-associated proteins, were retrieved from http://www.rcsb.org/ (Table S2), while the structural formulas of PHZ-OH was constructed using Chim3D (2010). Molecular docking between PHZ-OH and proteins was conducted by using AutoDock 4.0 and AutoDock Tools 1.5.6, in which independent docking calculation was conducted with 250,0000 evaluations using Lamarckian Genetic Algorithm. To validate the binding of PHZ-OH and AAK1 in physics, surface plasmon resonance was subsequently performed by using BIAcore4000 (BIAcore). The equilibrium-binding constant (KD, the lower value the higher affinity) of PHZ-OH and AAK1 was determined as described previously [24 (link)].