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TitleDeep mutational scan of a drug efflux pump reveals its structure-function landscape.
Journal, issue, pagesNat Chem Biol, Vol. 19, Issue 4, Page 440-450, Year 2023
Publish dateNov 28, 2022
AuthorsGianmarco Meier / Sujani Thavarasah / Kai Ehrenbolger / Cedric A J Hutter / Lea M Hürlimann / Jonas Barandun / Markus A Seeger /
PubMed AbstractDrug efflux is a common resistance mechanism found in bacteria and cancer cells, but studies providing comprehensive functional insights are scarce. In this study, we performed deep mutational ...Drug efflux is a common resistance mechanism found in bacteria and cancer cells, but studies providing comprehensive functional insights are scarce. In this study, we performed deep mutational scanning (DMS) on the bacterial ABC transporter EfrCD to determine the drug efflux activity profile of more than 1,430 single variants. These systematic measurements revealed that the introduction of negative charges at different locations within the large substrate binding pocket results in strongly increased efflux activity toward positively charged ethidium, whereas additional aromatic residues did not display the same effect. Data analysis in the context of an inward-facing cryogenic electron microscopy structure of EfrCD uncovered a high-affinity binding site, which releases bound drugs through a peristaltic transport mechanism as the transporter transits to its outward-facing conformation. Finally, we identified substitutions resulting in rapid Hoechst influx without affecting the efflux activity for ethidium and daunorubicin. Hence, single mutations can convert EfrCD into a drug-specific ABC importer.
External linksNat Chem Biol / PubMed:36443574 / PubMed Central
MethodsEM (single particle)
Resolution4.25 Å
Structure data

12816

7ocy

EMDB-12816, PDB-7ocy:
Enterococcus faecalis EfrCD in complex with a nanobody
Method: EM (single particle) / Resolution: 4.25 Å

Source
  • enterococcus faecalis (bacteria)
  • vicugna pacos (alpaca)
KeywordsMEMBRANE PROTEIN / ABC transporter / Nanobody / Enterococcus faecalis

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