Influence of the mutations on CR1 binding. Kinetic analyses were performed by SPR with recombinant soluble CR1 immobilized on the chip. The running buffer was composed of 10 mM HEPES (pH 7.4), 0.005% Tween-20, and 25 mM NaCl. The recombinant C3 proteins were injected for 300 seconds at 30 μL/min, followed by dissociation of 300 seconds. The chip was regenerated by injection of 0.5 M NaCl. The results are presented as a fraction of WT binding. (A) Association rate constant for CR1 binding. (B) Dissociation rate constant for CR1 binding. (C) Affinity for CR1 binding. (D) Example of SPR sensorograms and kinetic fit for MCP binding. (E) SPR analysis of the binding of C3d to CR1 in low ionic strength buffer. (F) C3b binding to CR1 in the same buffer. (G) Lack of binding of C3d to immobilized CR1 in normal ionic strength buffer. (H) The binding of C3b to immobilized CR1 in the same buffer. (I) Location of the putative CR1 binding site on C3b, comprising residues 727 to 767 of the α′NT region⁵  in blue and the suggested second binding site in the TED domain in a blue circle. Mutations are mapped in red. *P < .05; **P < .005, t test.