Abstract
Many pathogenic gram-negative bacteria have developed mechanisms to increase resistance to cationic antimicrobial peptides by modifying the lipid A moiety. One modification is the addition of phospho-ethano-lamine to lipid A by the enzyme phospho-ethano-lamine transferase (EptA). Previously we reported the structure of EptA from Neisseria, revealing a two-domain architecture consisting of a periplasmic facing soluble domain and a transmembrane domain, linked together by a bridging helix. Here, the conformational flexibility of EptA in different detergent environments is probed by solution scattering and intrinsic fluorescence-quenching studies. The solution scattering studies reveal the enzyme in a more compact state with the two domains positioned close together in an n-do-decyl-β-d-maltoside micelle environment and an open extended structure in an n-do-decyl-phospho-choline micelle environment. Intrinsic fluorescence quenching studies localize the domain movements to the bridging helix. These results provide important insights into substrate binding and the molecular mechanism of endotoxin modification by EptA.
Highlights
Pathogenic Gram-negative bacteria have evolved numerous mechanisms to evade the human immune system and developed widespread resistance to host-derived antimicrobials and traditional antibiotics
enzyme phosphoethanolamine transferase (EptA) purified in dodecyl--d-maltoside (DDM) micelles and DPC micelles were analysed by size-exclusion chromatography (SEC)-MALS in order to establish that the protein was monodisperse in different detergent micelles and amenable to Small-angle X-ray scattering (SAXS) studies
S1(a) and S1(b) of the supporting information display the overlayed chromatograms obtained from the UV absorption at 280 nm and refractive index (RI) signal superimposed on the right-angle scattering signal for EptA in DDM micelles and DPC micelles, respectively
Summary
Pathogenic Gram-negative bacteria have evolved numerous mechanisms to evade the human immune system and developed widespread resistance to host-derived antimicrobials and traditional antibiotics. EptA is found in a number of clinically relevant Gram-negative bacteria (Huang et al, 2018) and, given its important biological role in reducing the innate immune response, this enzyme has been a target for the development of potential inhibitors as therapeutic agents to treat multi-drug resistant bacterial infections (Kahler et al, 2018). Towards this aim, and in order to better understand the molecular mechanism involved in substrate binding and enzyme catalysis, the
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