PfRh4 interacts with the N-terminal 3 CCPs of CR1. (A) Schematic of CR1 polypeptide. Each box labeled 1 to 30 represents a complement control protein module (CCP) of 60-70 amino acid residues. The first 28 CCPs are organized based on homology into 4 long homologous repeats (LHRs) A-D, each consisting of 7 CCPs. The functional sites of CR1 are labeled site 1 and site 2. The recombinant CR1 fragments used in this study are indicated as black bars with relevant CCPs labeled above. NH2 indicates amino terminus; TM, transmembrane domain; and CYT, cytoplasmic tail. (B) Recombinant PfRh4 binds to CCP1-3. An ELISA was used to measure the interaction between CR1 fragments and rPfRh4. Microtiter wells were coated with CCP1-3, CCP10-11, CCP15-17, CCP21-22, or CCP15-25 at 0.5 μg/well. Recombinant PfRh4 was added at 0.5 μg/well. Bound PfRh4 was detected with an anti-PfRh4 monoclonal antibody 10C9. (C) Recombinant PfRh4 forms a complex with CCP1-3 but not CCP15-17. Immunoprecipitation experiments were performed in which combinations of recombinant proteins CCP1-3/rPfRh4 or CCP15-17/rPfRh4 were incubated with an anti-CR1 monoclonal antibody 1B4. For Western blot analyses of immunoprecipitated eluates, soluble CR1 fragments were detected with anti-CR1 monoclonal antibody 1B4 and rPfRh4 was detected with 10C9 monoclonal antibody. (D) Binding of sCR1 to rPfRh4 was inhibited by CCP1-3. Microtiter plates were coated with saturating concentrations of rPfRh4 (5 μg/well). CCP1-3 or CCP15-17 at concentrations of 0nM, 0.02nM, 0.23nM, 2.3nM, 23nM, or 234nM was incubated with sCR1 (23nM) before addition to wells. Interaction between sCR1 and PfRh4 was detected using anti-CR1 antibody HB8592, which detected sCR1 and not smaller CR1 fragments. ELISA experiments in panels B and D were repeated with similar results and their y-axis represent A_{405 nm} with error bars showing the range of duplicate readings.