Membrane lipid homeostasis dually regulates conformational transition of phosphoethanolamine transferase EptA

Abstract

The phosphoethanolamine transferase EptA utilizes phosphatidylethanolamine (PE) in the bacterial cell membrane to modify the structure of lipopolysaccharide, thereby conferring antimicrobial resistance on Gram-negative pathogens. Previous studies have indicated that excessive consumption of PE can disrupt the cell membrane, leading to cell death. This implies the presence of a regulatory mechanism for EptA catalysis to maintain a balance between antimicrobial resistance and bacterial growth. Through microsecond-scale all-atom molecular dynamics simulations, we demonstrate that membrane lipid homeostasis modulates the conformational transition and catalytic activation of EptA. The conformation of EptA oscillates between closed and open states, ensuring the precise spatiotemporal sequence of substrates binding. Interestingly, the conformation of EptA is significantly influenced by its surrounding lipid microenvironment, particularly the PE proportion in the membrane. PE-rich membrane conditions initiate and stabilize the open conformation of EptA through both orthosteric and allosteric effects. Importantly, the reaction mediated by EptA gradually depletes PE in the membrane, ultimately hindering its conformational transition and catalytic activation. These findings collectively establish a self-promoted model, illustrating the regulatory mechanism of EptA during the development of antibiotic resistance.

Fig. 1. Conformational transition of EptA.

a Simulation system for EptA in a bacterial cell membrane. The transmembrane and soluble domains are coloured blue and purple, respectively. The enzymatic active center is labelled. b The distances between H39 and N460 are calculated to indicate the process of the conformational transition of EptA. Source data are provided as a Source Data file. c Representative snapshots of EptA conformations in the membrane condition. The locations of H39 and N460 used for the distance calculations are labelled. d Free energy profile for the conformational transition process of EptA, along with representative structural snapshots (n  =  100 independent estimations; data are presented as mean values  ±  SD). Source data are provided as a Source Data file.

Fig. 2. Conformational transition of EptA creates distinct entry pathways for PE and LPS.

a Entry pathway for PE to access the active center of EptA in its closed conformation. b Interaction network between PE and the active site residues (orange sticks) of EptA. The PH2 and PH2’ helices are highlighted in blue. c Entry pathway for Re LPS to access the active center of EptA in its open conformation. d Interaction network between Re LPS and the active site residues (orange sticks) of EptA. The PH2 and PH2’ helices are highlighted in blue. e Cluster analysis of the interaction energy (IE) between EptA and PE, and between EptA and Re LPS. Data are shown as means. The color spectrum from yellow to purple indicates the increase in the intensity of IE. Source data are provided as a Source Data file.

Fig. 3. Interfacial interaction of EptA changes upon binding to PE.

a The hinge region of EptA exhibits distinct morphologies in the closed and open conformations. The contact residues are shown in a mesh model, with the transmembrane and soluble domains presented in blue and purple, respectively. b Electrostatic interactions between the transmembrane and soluble domains vary across different conformations of EptA. The interactions are shown as dashed lines. c Evolution of the electrostatic interaction during the simulations of EptA opening. Source data are provided as a Source Data file. d Interactions between bound PE and interfacial residues. The hydrogen bonds are shown in dashed lines. e The distances between H39 and N460 in both the pure EptA system and the EptA-PE complex system during the simulations. Source data are provided as a Source Data file.

Fig. 4. Interaction between EptA and the membrane regulates the stability of the EptA open conformation.

a Spatial positions of acidic residues (red spheres) on the dorsal region of EptA soluble domain in the closed (left) and open (right) conformations. b Surface potentials of EptA, shown from both front and side views. Red surface indicates negative potential; blue surface indicates positive potential. c Interaction energy between acidic residues and PE and PG in the cell membrane. Data are shown as means. Source data are provided as a Source Data file. d Sequence profile of 7308 EptA protein sequences indicates the evolutionary conservation of the acidic residues involved in interactions with PE. e Spatial locations of residues D309, E317, and D370, subjected to computational mutational analysis. f Conformational distribution of EptA mutants calculated based on three simulation replicates. Source data are provided as a Source Data file.

Fig. 5. Conformational dynamics of EptA in membranes with various proportions of PE.

A distance less than 2 nm is used as a cutoff to signify the closed conformation. Source data are provided as a Source Data file.

Fig. 6. Experimental mutations of key residues affect the function of MCR-1.

a Electrostatic residues in the hinge region and acidic residues in the dorsal region of the soluble domain. b Minimum inhibitory concentrations of colistin against E. coli DH5α strains expressing wild-type and MCR-1 mutants.

Fig. 7. Different EptA proteins demonstrate a consistent conformational regulatory mechanism.

a An unrooted evolutionary tree for the protein sequences of EptA from 160 species, Specifically, HpEptA, NmEptA, and SeEptA belong to Cluster 1 (brown), Cluster 2 (blue), and Cluster 3 (orange), respectively. b Sequence profiles of the residues in the EptA hinge region for the three clusters. c Sequence profiles of the residues in the EptA dorsal region of the soluble domain for three clusters. d Conformational dynamics of EptA from HpEptA, NmEptA, and SeEptA in the simulations. Source data are provided as a Source Data file. e Interaction energy of within the hinge regions of HpEptA, NmEptA, and SeEptA. Hydrophobic interactions are shown in green, while polar interactions are shown in yellow (n  =  2001 independent estimations; data are presented as mean values  ±  SD). Source data are provided as a Source Data file. f Interaction energy between the membrane and the soluble domains of HpEptA, NmEptA, and SeEptA (n  =  5001 independent estimations; data are presented as mean values  ±  SD). Source data are provided as a Source Data file.

Fig. 8. A schematic illustrating the relationship between the conformational transition of EptA and the lipid microenvironment in the membrane.

The conformational transition of EptA is modulated by its local membrane environment, especially the abundance of PE. The head groups of PE molecules are highlighted in yellow.