High Resolution Cryo-Em Structure of the Pseudomonas Aeruginosa OprM Multidrug Efflux Pump Provides Target for Rational Inhibitor Design

OprM is the outer membrane channel component of resistance-nodulation-division (RND) efflux pumps in Pseudomonas aeruginosa, a high-priority human pathogen. OprM-dependent pumps play diverse roles in bacterial physiology and mediate resistance to critical antibiotic classes, including fluoroquinolones, aminoglycosides, and broad-spectrum β-lactam/β-lactamase inhibitor combinations such as ceftazidime/avibactam. As the outer membrane component of the tripartite efflux system, OprM gates substrate export through an iris-like periplasmic pore that opens upon association with its inner membrane partners. Here we present a single-particle cryo-EM structure of OprM at 2.08 Å, the highest resolution OprM structure to date. The structure reveals a closed leucine-lined periplasmic pore, consistent with previous X-ray structures. A detailed structure-based comparison of the closed and open states reveals the conformational changes accompanying periplasmic pore opening, including iris-like twisting of the coiled-coil helices, displacement of the gating Leu429 residues, and a broader reorganization of salt bridges than previously described. Conservation of the MexA-binding interface across multiple Mex proteins suggests a shared mechanism of OprM engagement. Three lipopolysaccharide (LPS) molecules are resolved at the β-barrel interface, bridging adjacent OprM monomers through a combination of hydrophobic interactions with the fatty acyl chains and specific polar contacts with the KDO, heptose, and phosphate groups. This binding mode resembles that of other generic β-barrels and is expected to be preserved in vivo. Together, these findings provide the most detailed structural characterization of OprM to date, offering new insights into its gating mechanism and membrane interactions with implications for the development of efflux pump inhibitors.