Interaction between CR3-α_MI and C3-derived opsonins in physiologic orientation. (A) Schematic overview of surface opsonization for the SPR study. C3b was covalently deposited via its thioester domain by on-chip formation of the C3 convertase and injection of C3. The resulting C3b surfaces were either left unchanged or converted to iC3b and C3dg by injecting factor I (FI) with cofactors factor H or soluble CR1, respectively (supplemental Methods; supplemental Figure 2A). (B-C) Interaction profile of CR3-α_MI toward C3b, iC3b, and C3dg. Recombinant CR3-α_MI (0.03-2 µM) was injected over all 3 opsonin surfaces in Mg²⁺-containing buffer, and the interaction was recorded as change in SPR response in resonance units (RU). In the case of iC3b and C3dg, processed responses (C; blue) were fit to a 1:1 Langmuir model (C; red) to extract kinetic rate constant and binding affinity (B); the C3b surface did not result in a significant binding response. The binding data are representative of 2 independent experiments with comparable results. (D-F) Evaluation of binding specificity of the CR3-α_MI interaction with C3dg. (D) Metal ion dependence was tested by injecting CR3-α_MI (500 nM) to C3dg in the presence of either 1 mM MgCl₂ or 3 mM EDTA. Binding sites were validated using blocking antibodies against (E) the α_MI domain of CR3 (mAb CBRM1/5) and (F) the C3d domain of C3 (mAb 1149).