Fig. 2. Binding of the Wuhan-1 reference strain Spike trimer and monomeric RBD to ACE2.
a Spike trimer in complex with one or two ACE2 dimers (indicated by red arrows). b AFM image (left) and simulation using MDS modeling (right) of Spike trimer bound to one ACE2. c Schematic illustration of single-molecule force spectroscopy experiments, in which dissociation forces between the Spike trimer (or RBD) on AFM tips and membrane-anchored ACE2 expressed on VeroE6 cells were measured. d Exemplary force–distance curves from the experiments. The Spike trimer (green lines) exhibits up to three interactions per force curve. Multiple bonds either break sequentially ((2) and (3)) or simultaneously ((II) and (III)). Monomeric RBD shows only one interaction per force curve (red line). e Experimental probability density functions (PDF) at a fixed pulling velocity. Force maxima for the Spike trimer (green curve) correspond to the simultaneous dissociation of one (I) two (II) or three (III) bonds. Monomeric RBD (red curve) shows only one force maximum, reflecting the dissociation of a single bond. f Dynamic force spectroscopy from experiments at different pulling speeds. Dependence of dissociation forces on force loading rate. Data of the first peaks were fitted with the Bell–Evans model (thin green line), which assumes that a sharp single energy barrier is crossed for dissociation and yields the kinetic off rate, koff, and the dissociation path length, xB (Table 1). Using the fitting parameter of the Bell–Evans theory, the orange line and blue line were calculated using the Markov binding model for uncorrelated failure of two and three bonds, respectively. The thick magenta line is the Bell–Evans fit of raw force data for RBD. g Dependence of binding activity on preset contact (interaction) time between the AFM tip and cellular surfaces for a Spike trimer (green circles) and for a single RBD (pink circles). Binding activity ratios for two (orange diamonds) and three (blue triangles) bonds of Spike trimer are also shown. Kinetic on-rates are retrieved from exponential fittings using pseudo-first-order kinetics of a bimolecular reaction (solid lines). Source data are provided as a Source Data file.