28vj

NorA bound to miniprotein I-23

spinqualitylabelsall models 28vj, resolution 2.79Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 28vj is a 2 chain structure with sequence from Escherichia coli, Staphylococcus aureus and Synthetic construct. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: Electron Microscopy, Resolution 2.79Å Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Publication Abstract from PubMed Multidrug efflux pumps transport antibiotics across the cellular membrane resulting in resistance conferred to the host organism. Efflux pump inhibitors (EPIs) potentiate the efficacy of antibiotics by blocking drug efflux and hold promise as adjuvant therapeutics in the fight against multidrug resistant pathogenic bacteria. A hurdle in the field has been the lack of selectivity of small molecule EPIs which often display off-target toxicity due to non-specific binding. To tackle this specificity challenge, we aimed to maximize an inhibitor's binding surface area to efflux pumps by designing miniprotein EPIs using computational protein design and an E. coli co-expression assay to screen inhibition in cells. We used S. aureus NorA as a model efflux transporter since it confers drug resistance to fluoroquinolones, puromycin, and other cytotoxic compounds. Starting from a focused miniprotein library of only 86 members, we identified inhibitors in the screen that blocked NorA transport under active efflux conditions in vitro. Our most promising inhibitor I-23 was validated by solving a cryo-EM structure of the miniprotein in complex with NorA, which stabilized the transporter in the outward-open conformation. I-23 has a ferredoxin-like fold with one of its beta-hairpins inserted into the substrate binding pocket of NorA and other parts of the globular fold occupying the shallow pocket and making extensive intermolecular contacts with NorA. An arginine residue on the tip of the hairpin loop was positioned near an anionic patch required for NorA antibiotic efflux. The identified structural motifs in this work could be employed to explore the molecular properties of peptidoglycan penetration; full realization of the therapeutic potential of the designed miniprotein inhibitors will require determining the principles for facilitating passage of ~7 to 8 kDa miniproteins across the peptidoglycan bacterial cell wall. Miniprotein inhibitors of the Staphylococcus aureus efflux transporter NorA.,Mishra P, Chazin-Gray A, Lamon G, Kim D, Baker D, Traaseth NJ bioRxiv [Preprint]. 2026 Mar 5:2026.03.05.709893. doi: , 10.64898/2026.03.05.709893. PMID:41847033 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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