In vitro characterization of the colibactin-activating peptidase ClbP enables development of a fluorogenic activity probe

Abstract

The gut bacterial genotoxin colibactin has been linked to the development of colorectal cancer. In the final stages of colibactin's biosynthesis, an inactive precursor (precolibactin) undergoes proteolytic cleavage by ClbP, an unusual inner-membrane-bound periplasmic peptidase, to generate the active genotoxin. This enzyme presents an opportunity to monitor and modulate colibactin biosynthesis, but its active form has not been studied in vitro and limited tools exist to measure its activity. Here, we describe the in vitro biochemical characterization of catalytically active, full-length ClbP. We elucidate its substrate preferences and use this information to develop a fluorogenic activity probe. This tool will enable the discovery of ClbP inhibitors and streamline identification of colibactinproducing bacteria.

Medicinal plant extracts protect epithelial cells from infection and DNA damage caused by colibactin‐producing Escherichia coli , and inhibit the growth of bacteria

“…This has resulted in the development of a fluorogenic activity probe as a ClbP inhibitor for the inhibition of colibactin production (Volpe et al . 2019). Moreover, antibiotics administered for the treatment of ExPEC strain infection include β‐lactams, fluoroquinolones, aminoglycosides and trimethoprim‐sulfamethoxazole.…”

Structural basis of colibactin activation by the ClbP peptidase

“…ClbP residues S95, K98 and Y186 are indispensable for enzymatic activity, and, in a crystal structure of the periplasmic domain, these residues converge to form the catalytic site 19 . ClbP cleaves precolibactin analogs with varied acyl chains and amide substituents but is highly specific toward the d-asparagine sidechain 20 . In the absence of substrate-bound structures, the mechanism underlying the substrate specificity of ClbP is not yet known.…”

“…Because molecules containing the myristoyl substituent found in precolibactin are poorly soluble, we used an analog that replaces the myristoyl chain with a 4-phenylbutyryl group (compound 1, N-4-(4-bromophenyl)butanoyl-d-asparaginyl-l-alanine methyl ester; Fig. 2a), which is more soluble and is processed as effectively as myristoylated analogs 20 . We introduced a bromine substituent at the para position of the phenyl ring as an anomalous scatterer to validate the presence of the substrate analog in the electron density maps and aid in model building.…”

“…We explored the role of interactions between the prodrug motif and its binding site on ClbP's enzymatic function by measuring cleavage of a fluorogenic substrate analog by purified ClbP variants (Extended Data Fig. 3b,c) 20 . TM2-TM3 linker mutation F462A did not affect enzyme activity, whereas the W466A mutation completely abrogated activity, demonstrating that W466 is essential for peptidase activity in vitro (Fig.…”

“…We hypothesized that N331 enforces the specificity of ClbP for substrates with d-asparagine prodrug motifs and that its sidechain orientation, set by a hydrogen bond with E92 to present a hydrogen bond-accepting carbonyl to the substrate, is responsible for excluding d-aspartate analogs, which are cleaved at marginal rates by ClbP (ref. 20 ). To test this hypothesis, we perturbed the interaction network determining the N331 orientation (Fig.…”