Structural and mechanistic insights into polymyxin resistance mediated by EptC originating from Escherichia coli.

Gram-negative bacteria defend against the toxicity of polymyxins by modifying their outer membrane lipopolysaccharide (LPS). This modification mainly occurs through the addition of cationic molecules such as phosphoethanolamine (PEA). EcEptC is a PEA transferase from Escherichia coli (E.coli). However, unlike its homologs CjEptC (Campylobacter jejuni) and MCR-1, EcEptC is unable to mediate polymyxin resistance when overexpressed in E.coli. Here, we report crystal structures of the C-terminal putative catalytic domain (EcEptCΔN, 205-577 aa) of EcEptC in apo and Zn2+ -bound states at 2.10 and 2.60 Å, respectively. EcEptCΔN is arranged into an α-β-α fold and equipped with the zinc ion in a conserved mode. Coupled with isothermal titration calorimetry (ITC) data, we provide insights into the mechanism by which EcEptC recognizes Zn2+ . Furthermore, structure comparison analysis indicated that disulfide bonds, which play a key role in polymyxin resistance, were absent in EcEptCΔN. Supported by structural and biochemical evidence, we reveal mechanistic implications for disulfide bonds in PEA transferase-mediated polymyxin resistance. Significantly, because the structural effects exhibited by disulfide bonds are absent in EcEptC, it is impossible for this protein to participate in polymyxin resistance in E.coli. This article is protected by copyright. All rights reserved.