Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-d-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads.

Colibactin 是一种由肠道细菌产生的 DNA 交联剂，与结直肠癌有关。其生物合成采用前体药物抗性机制：在细胞质中组装的无毒前体在输出到周质后被激活。这种激活是由 ClbP 介导的，ClbP 是一种内膜肽酶，具有 N 端周质催化结构域和 C 端三螺旋跨膜结构域。尽管跨膜结构域是大肠杆菌素激活所必需的，但其在催化中的作用尚不清楚。我们的全长 ClbP 与产物类似物结合的结构揭示了对底物结合和酶稳定性和相互作用很重要的域间界面，这解释了 ClbP 对N-酰基-d-天冬酰胺前药基序的选择性。基于结构和生化证据，我们提出ClbP二聚化形成一个扩展的底物结合位点，该位点可以容纳假二聚体precolibactin及其在两个ClbP活性位点中的两个末端前药基序，从而能够协调激活两个亲电弹头。