A “Drug Sweeping” State of the TriABC Triclosan Efflux Pump from Pseudomonas aeruginosa

• Published in: Structure (London) 2021-03, Vol.29 (3), p.261-274.e6
• Main Authors: Fabre, Lucien, Ntreh, Abigail T., Yazidi, Amira, Leus, Inga V., Weeks, Jon W., Bhattacharyya, Sudipta, Ruickoldt, Jakob, Rouiller, Isabelle, Zgurskaya, Helen I., Sygusch, Jurgen
• Format: Article
• Language: English
• Subjects: membrane protein structure; periplasmic membrane fusion protein; Pseudomonas aeruginosa; resistance-nodulation-division superfamily; RND inner membrane transporter; single-particle cryo-EM; substrate efflux tunnel; substrate translocation; TriABC; triclosan/SDS-specific efflux pump
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2020.09.001

The structure of the TriABC inner membrane component of the triclosan/SDS-specific efflux pump from Pseudomonas aeruginosa was determined by cryoelectron microscopy to 4.5 Å resolution. The complete structure of the inner membrane transporter TriC of the resistance-nodulation-division (RND) superfamily was solved, including a partial structure of the fused periplasmic membrane fusion subunits, TriA and TriB. The substrate-free conformation of TriABC represents an intermediate step in efflux complex assembly before the engagement of the outer membrane channel. Structural analysis identified a tunnel network whose constriction impedes substrate efflux, indicating inhibition of TriABC in the unengaged state. Blind docking studies revealed binding to TriC at the same loci by substrates and bulkier non-substrates. Together with functional analyses, we propose that selective substrate translocation involves conformational gating at the tunnel narrowing that, together with conformational ordering of TriA and TriB, creates an engaged state capable of mediating substrate efflux. [Display omitted] •Cryo-EM structure of substrate-free TriABC at 4.5 Å resolution•Periplasmic membrane fusion subunits are partially ordered•An arginine controls the pKa of surrounding aspartate residues to donate protons•Loop motion gates substrate tunnel opening, controlling substrate specificity TriABC is a triclosan/SDS-specific efflux pump from Pseudomonas aeruginosa linked to biocide resistance. Substrate efflux depends on a tunnel in TriC subunits, supported by functional and docking data. Tunnel narrowing by an internal gating loop situated after Val 698 inhibits efflux. Subunits TriA and TriB show disorder in the absence of substrate.