Fig. 3: Probing the kinetic and thermodynamic parameters underlying PMX53 antagonist binding to C5aR.

a Schematic representation of an AFM tip tethered with the high-affinity PMX53 antagonist probed against C5aR. Height (b) and adhesion (c) maps recorded while probing C5aR embedded in the lipid bilayer with a PMX53 modified AFM tip. d The interaction between PMX53 and C5aR was probed over a wide range of LRs by variating the retraction speed in the force–distance curves. Low LRs were explored at 500 nm s⁻¹ and 2 µm s⁻¹ pulling speeds, while high LRs were reached at 50 µm s⁻¹ pulling speed. e Extracting the parameters describing the PMX53–C5aR free energy landscape. A ligand–receptor bond can be described using a simple two-state model, where the bound state resides in an energy valley and is separated from the unbound state by an energy barrier. The transition state must overcome an energy barrier to separate ligand and receptor. τ⁻¹(F) and τ⁻¹(0) are residence times linked to the transition rates for crossing the energy barrier under an applied force F and at zero force, respectively. ΔG_bu is the free-energy difference between bound and unbound state. f Force–volume (FV)-AFM and FD-based AFM were used to explore binding at low and high LRs, respectively. For each pixel of the topography, the tip is approached and retracted using a linear (FV-AFM) or oscillating movement (FD-based AFM). g A force–distance curve (upper panel) can be displayed as a force–time curve (bottom panel), from which the loading rate can be extracted via the slope of the curve just before bond rupture. Probing the kinetic and thermodynamic parameters underlying PMX53 antagonist binding to C5aR (h) and and two mutants, (i) C5aR^R175V/Y258V and (j) C5aR^D282A. Fitting the data using the Friddle–Noy–de Yoreo model (thin green lines) provides average F_eq, ΔG_bu, and residence time (τ_0.5) values with errors representing the s.e.m. Each circle represents one measurement. Darker green shaded areas represent 99% confidence intervals, and lighter green shaded areas represent 99% of prediction intervals. A reduction of the affinity WT > R175V/Y258V > D282A is observed. For each condition, data are representative of at least three independent experiments.