Analysis of gC1qR binding to HKD5. (A) The recombinant HKD5 construct boundaries are shown with residue numbers indicated for D5-1, D5-2, and shorter peptides used in the ITC and gel filtration experiments. (B) Gel filtration of HKD5 combined with gC1qR at different molar ratios. Elution profiles are shown on the left, and Coomassie-stained SDS-PAGE gels of the fractions collected are shown on the right. The 3:1 ratio of HKD5 to gC1qR reveals excess HKD5 eluting separately suggesting the trimer only supports 2 HKD5 polypeptides. (C) ITC measurements of gC1qR binding to HKD5. D5 was titrated into gC1qR in the presence of Zn²⁺ or EDTA. This was fit to a 3-site sequential binding model with no difference between curves produced in the presence or absence of Zn²⁺. (D) Gel filtration of D5-1, D5-2, and gC1qR in the presence of 50 μM ZnCl₂ (black) or 5 mM EDTA (red) revealing D5-1 has a Zn²⁺ dependence whereas HKD5-2 does not. (E) ITC experiments with D5-1 and D5-2 respectively titrated into gC1qR in the presence of Zn²⁺. Similarly to full-length HKD5, D5-1 was fitted to a 3-site sequential binding model, and binding was Zn²⁺ dependent. (F) The binding of D5-2 was Zn²⁺-independent and was fit to a single-site binding model with a calculated N = 2. (G) HKD5 derived shorter peptides titrated into gC1qR. All titrations excluding HK 493-516 were performed in the presence of Zn²⁺. HK 493-516 was the only peptide to show binding, and the curve resulted in binding affinities and N values comparable to D5-2.