Fig. 3.

Identification of serine protease active-site residues at the DLD homodimer interface. (A) One subunit of human DLD homodimer (1ZMC) is shown in ribbon representation with the dimer interface domain (residues 350–474) in green, and residues S456, H450, and E431 are shown as sticks. The figure created with PyMOL. (B) The C-term, corresponding to the dimer interface domain, was expressed in E. coli and purified (SI Methods and SI Table 4). C-term (5 μg) was analyzed by SDS/12% PAGE and SYPRO orange staining alongside the full-length human DLD. (C) Aliquots (5 or 10 μg) of C-term DLD were incubated with or without 1 mM diisopropyl fluorophosphate or phosphorofluoridate (DFP) in duplicate for 24 h at 37°C. All protein samples were resolved on SDS/12% PAGE, visualized by silver staining, eluted from gel slices by passive diffusion, and tested for proteolytic activity with m-fxn substrate as described in Fig. 1 C and D. Shown are the results obtained with eight independent gel slices. (D) WT and mutant DLD proteins were purified at the same time and assayed simultaneously in a 96-well plate by using a fluorogenic peptide substrate consisting of 13 aa from the m-fxn N-terminal region, with a fluorescent donor and a quenching acceptor (see Experimental Methods). Each reaction contained 2 μM DLD and 50 μM peptide in 100 μl of 10 mM Tris·HCl, pH 8.0, and 50 mM NaCl. Total fluorescence was measured after 2, 4, and 8 h of incubation at 37°C. Blanks containing DLD protein only or substrate only in buffer were run in parallel with proteolytic reactions. Bars represent fluorescence intensity after background subtraction. Each bar represents the mean ± SE of three (WT and D444V) or two (S456A, S456A/D444V, E431A, and H450A) independent experiments, each conducted in duplicate. SDS/PAGE analysis of all proteins is shown in SI Fig. 8A.