Mapping of the gC1qR-binding site for HKD5. (A-C) ITC experiments with HKD5, D5-1, and D5-2, respectively titrated against the gC1qR variants compared with wt-gC1qR. Deletion of the gC1qR G3-loop (gC1qRdelG3) in blue showed no binding to D5-1 and reduced binding to both HKD5 and D5-2, whereas the other variants were comparable to wt-gC1qR. (D) The HKD5-binding site maps to the region of the G3-loop (blue) that forms the boundary of a pocket (G3, shown as blue dashed ellipse), which extends across the β-sheet to the Zn²⁺ site (gray sphere). The locations of the G1 pocket (purple dashed ellipse), G2-loop (orange), and G1-loop (red) are shown. (E) A schematic representation of the proposed D5 binding to gC1qR. The ligand-free gC1qR and HKD5 are shown top left. D5-1 is represented as a larger circle (blue) connected to a smaller circle representing D5-2 (red). The larger size of the D5-1 circle is representative that this region binding to gC1qR cannot be emulated by short peptides. gC1qR is shown in gray to represent the Zn²⁺-free form, and flexible anionic loops are shown colored as in panel D with the G1- and G2-loops radially located and the G3-loop in the center. The binding of D5-1 is sequential whereby tight binding of the first D5-1 (K_D1) is followed by subsequently reduced affinity second and third binding events (K_D2 and K_D3), suggesting a third HKD5 binding is sterically occluded (shown as transparent). The binding of D5-2 to gC1qR is not sequential, and all binding events to gC1qR have equivalent affinity (K_D1).