Fig. 1: Development and characterization of an ASCPYD-specific protein binder.
A Structure of ASC comprising PYD and CARD (PDB ID: 2KN6, 3J63). ASCs are oligomerized through homotypic interaction between PYDs. The surface model with gray color represents oligomerized ASCPYD molecules. B Structural representation of the repebody scaffold (PDB ID: 3RFS) used for the development of an ASCPYD-specific protein binder. A repebody library was constructed by randomizing variable sites on LRR modules on a concave surface, followed by phage display selection against ASCPYD. Mutation sites for library construction are shown in color: first library sites in purple, second library sites in green, and additional mutation sites in cyan. C Relative binding affinities of an initially selected repebody (r5H) and affinity-matured repebodies against ASCPYD using ELISA. The highest binding intensity was observed by rB7. The data represent the means ± SDs from triplicate experiments. D Titration curve of rB7 against ASCPYD using ITC. The binding affinity (KD) of rB7 for ASCPYD was determined to be 1.5 nM. Data were representative of three independent experiments. E Specificity of rB7. The binding of myc-tagged rB7 (4 μM) was tested against various proteins (50 nM) coated on immunoplate. BSA was used as a negative control. Relative binding was quantified by ELISA using myc-tag antibody. The data represent the means ± SDs from triplicate experiments. ****p < 0.0001 compared with the control (two-tailed unpaired Student’s t-test). F Elution profile of rB7 in complex with ASCPYD on size-exclusion chromatography. Inset represents SDS/PAGE analysis for eluted fractions from size-exclusion chromatography.
