Fig. 3. Thermodynamical characterization of the interactions between mNgR1 and either reovirus T1L or single μ1σ3 heterohexamers, and predicting the mNgR1–σ3 binding interface. (A) Representation of the experimental setup, with an AFM tip functionalized with a reovirus T1L virion and mNgR1 proteins grafted on a gold-coated surface, operated in FV mode. (B) Box plot of the BP calculated for the T1L–mNgR1 interaction, with (control) and without addition of EDTA (5 mM). (C) DFS plot showing the distribution of rupture forces as a function of their LR, measured between T1L virion and mNgR1. Binding probability is plotted (as inset) as a function of the contact time. (D) Representation of the experimental setup, with an AFM tip functionalized with mNgR1 and single σ3μ1 heterohexamers grafted on a gold-coated surface, operated in FV mode. (E) Box plot of the BP calculated for the mNgR1-single heterohexamers, with (control) and without addition of EDTA (5 mM). (F) DFS plot showing the distribution of rupture forces as a function of their LR, measured between mNgR1 and single heterohexamers. For all DFS plots, the solid line represents the fit of the data with the Bell–Evans fit (for simple ligand–receptor bond). For contact time plots, data points represent mean BP calculated for each contact time and were fitted using a least-squares fit of a monoexponential decay. For all DFS and contact time plots, the error bars indicate SD. For all BP plots, one data point represents the BP obtained for one map of 1024 FD curves. The horizontal line within the box indicates the median, boundaries of the box indicate the SEM, and the whiskers indicate the SD. All data are representative of at least n = 3 independent experiments. (G) Structural prediction of the complex between mNgR1 (in orange surface) and the two σ3A (in light-grey surface) and σ3B (in dark-grey surface) subunits. (H) RMSD of indicated chains computed using mNgR1 as the reference for the MD trajectory alignment. The plots are colored in orange, light grey, and dark grey for mNgR1, σ3A and σ3B, respectively. R1, R2, and R3 denote MD replicates.
