9HCI

structure of the double Cys-substituted cross-linked AcrB variant S562C_T837C

Summary for 9HCI

• Entry DOI: 10.2210/pdb9hci/pdb
• Descriptor: Multidrug efflux pump subunit AcrB, nonane, SULFATE ION, ... (12 entities in total)
• Functional Keywords: rnd multidrug efflux pump, antimicrobial resistance, intermediate conformational state, crosslinked structure, transport protein
• Biological source: Escherichia coli K-12; More
• Total number of polymer chains: 5
• Total formula weight: 389328.32

Authors

Brandstaetter, L.,Mueller, R.T.,Pos, K.M. (deposition date: 2024-11-10, release date: 2025-11-19, Last modification date: 2026-05-20)

Primary citation

Bornsen, C.,Muller, R.T.,Vieira Da Cruz, A.,Jimenez-Castellanos, J.C.,Meurillon, V.,Brandstatter, L.,Lodinsky, E.V.,Athar, M.,Vargiu, A.V.,Hartkoorn, R.C.,Flipo, M.,Frangakis, A.S.,Pos, K.M.
Molecular mechanism of transition-state inhibitors of bacterial antibiotic efflux pumps.
NPJ Antimicrob Resist, 4:-, 2026

PubMed Abstract: In many Gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae, the AcrAB-TolC efflux pump is central to multidrug resistance. We report the development of BDM91531, a nanomolar pyridylpiperazine inhibitor that potentiates the activity of several antibiotics. Structural analyses by X-ray crystallography and cryo-EM revealed that the divalent cationic BDM91531 binds AcrB through electrostatic interactions with a central role for residues D408 and E947, trapping protomers in an O to L transitional state and blocking the conformational cycling of the trimer. Differential scanning fluorimetry and susceptibility tests confirmed this inhibitory mechanism. Negative charges at the cytoplasmic rim are essential for inhibitor uptake as electrostatic attraction from rim carboxylates, including E947 and D951, facilitates entry. Loss of D951 abolished inhibitor sensitivity, whereas introducing alternative negative charges restored activity. These findings establish BDM91531 as a potent AcrB efflux pump inhibitor and highlight structural determinants for inhibitor access and binding.

PubMed: 42082832
DOI: 10.1038/s44259-026-00207-6

Experimental method

X-RAY DIFFRACTION (2.6 Å)