Fig. 4.

Design chimeric QTY receptors. (A) Schematic of the chimera design. CXCR4^QTY is green. CCR5^QTY’s 7TM regions (yellow) and IC loops were chosen as the backbone of the design (protein sequences are in SI Appendix). In Chimera A (Bottom Left) (CCR5^QTY-N-3(GS)n) the 3 EC loops of CCR5^QTY were replaced by GS linker sequence (red) with the same length (Upper, yellow loops), but CCR5^QTY N terminus (red line) was unchanged. In Chimera B (Bottom Right) (CCR5^QTY-7TM: CXCR4^QTY-N,3ECL), the N terminus and EC loops of CCR5^QTY were replaced by N terminus and 3 EC loops of CXCR4^QTY (green N-terminal and 3 green loops). (B) Three independent T_m measurements by NanoDSF to evaluate the thermostability. CCR5^QTY: 73.3 ± 0.9 °C, Chimera A: 68.2 ± 1.8 °C, Chimera B: 68.3 ± 1.8 °C. (C and D) MST ligand-binding measurement for Chimera A and Chimera B. (C) Chimera B binds both CXCL12 and CCL5 with affinity of 54.7 ± 19.6 nM and 150 ± 45 nM, respectively. (D) Chimera A binds CCL5 with affinity of 64.4 ± 40.8 nM. These results suggest that the N terminus and 3 EC loops are the most crucial regions for the ligand-binding activities of these chemokine receptors.