FIGURE 3.

“Velcro” engineering of human CD47. A, schematic of strategy to increase CD47 affinity for SIRPα by N-terminal and loop extension. The co-crystal structure of SIRPα (green in surface representation) and CD47 (magenta in cartoon representation) is highlighted with the N-terminal pyroglutamate (blue) and the loop at positions 52–54 (cyan) regions in sphere representation. The arrows represent the direction of intended amino acid extension. B, extension library design. Residues colored in pale orange are native to CD47 and are included in randomization. Residues colored in blue indicate the three additional amino acids that were added for N-terminal extension, and residues colored in cyan represent the 0, 2, or 4 additional amino acids that were added for loop extension. NxLy represents the types of molecule present in the extension library, where x and y indicate the number of amino acid extensions at the N terminus and at the FG loop region, respectively. The randomized DNA of N3L0, N3L2, and N3L4 was made separately and then mixed to transform a single yeast library. AA, amino acid.