Fig. 2: Multiplex probing of C5aR intra- and extracellular binding sites as a method discriminate C5aR orientation within lipid membrane.

Two different AFM tip chemistries were used to target either the His₆-tag C-terminal end of C5aR using tris-NTA–Ni²⁺ functionalized AFM tips (a–d) or the N-terminal end of C5aR using the endogenous C5a ligand (e–h). AFM height and adhesion images were recorded over the same lipid patch with a tris-NTA–Ni²⁺ tip (b) and (c) and a C5a ligand tip (f) and (h). d, h Representative FD curves showing either specific adhesion events (curves 1, 2) or no/unspecific interactions (curves 3, 4) were extracted from the adhesion maps in (c) and (g). 2D histograms of force vs. height for tris-NTA–Ni²⁺ modified tips (i) and C5a tips (j). k Height distribution of the receptors interacting with the tris-NTA–Ni²⁺ or the C5a tip. Two populations can be clearly distinguished, one below 1.75 nm in height, where C5a tips mostly interact with the extracellular side of C5aR, and another one above 3.5 nm in height, where tris-NTA–Ni²⁺ functionalized tips interact with the intracellular side of C5aR. l Height map overlay of the region marked by a white square in (f) and corresponding specific adhesion events extracted from the same areas in the maps in (c) and (g). Adhesion events between the C5a ligand and the N-terminal side of C5aR are shown as red dots, while the events rising from the tris-NTA–Ni²⁺ AFM tip interaction with the His₆-tagged C-terminal side of C5aR are displayed as blue dots. White dotted circles mark receptors with a height less than 1.75 nm, where the C5a ligand and the N-terminal side of C5aR interact. The overlay image shows how the orientation of single C5aR particles can be identified using our multiplex probing method. Data are representative of at least three independent experiments. Data in (k) are displayed as mean ± S.D. and the ANOVA one-way Tukey test was used to report the statistical significance.