Preparation of WCR-YDA

All constructs of D4 WT, D434A, and YDA were subcloned into the pET-30a vector with an N-terminal His₆-tag, and proteins were expressed in E. coli BL21 RIL codon plus cells (Stratagene) and purified using the Ni-NTA agarose affinity resin (Marvelgent) as described previously (19). For preparation of WCR-YDA, the YDA-bound resin was resuspended with WCR (1:10 molar ratio) in 5 ml of labeling buffer [50 mM Tris, pH 8.05, containing 150 mM NaCl, 20 mM imidazole, 50 mM arginine, 50 mM glutamate, and 1 mM Tris(2-carboxyethyl)phosphine (TCEP)] and the mixture was gently shaken for 2 h at room temperature, or at 4°C overnight in a gyratory shaker. WCR-YDA was then washed with 50 ml of the wash buffer (80 mM Tris, pH 7.9, 300 mM NaCl, 40 mM imidazole) containing 4% (v/v) dimethyl sulfoxide and then with 300 ml of the wash buffer. WCR-YDA was eluted from the resin with the elution buffer (50 mM Tris, pH 7.9, 300 mM NaCl, 300 mM imidazole). Collected fractions were concentrated in an Amicon Ultra 0.5 ml Centrifugal Filter (Millipore), and the buffer solution was exchanged to 20 mM Tris, pH 7.4, 160 mM NaCl. The protein concentration of the WCR-YDA solution was determined by the Bradford assay. All steps were performed at 4°C unless otherwise mentioned.