Fig. 2. The prodrug motif binds at the interface between periplasmic and transmembrane domains.
a, Substrate analog included in crystallization of catalytically inactive ClbP. Our data suggest that this molecule is hydrolyzed during crystallization, as the atoms in gray are not observed in the electron density map. b, Two views, related by a 90° rotation, of the hydrolysis product bound at the active site. The d-asparagine sidechain of the prodrug motif interacts with periplasmic domain residues S188, H257 and N331, and the acyl chain interacts with TM2–TM3 linker residues F462 and W466 (sidechains shown as sticks). Polder map omitting the product contoured at 7σ is colored in cyan, and bromine anomalous difference Fourier map contoured at 3.5σ is colored in purple. c, Enzymatic activity of purified ClbP variants measured as cleavage of a fluorogenic substrate analog (Extended Data Fig. 3c). The plot represents triplicate measurements normalized to the average for wild-type (WT) ClbP.