Deep mutational scan of a drug efflux pump reveals its structure-function landscape

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Drug efflux is a primary mechanism of resistance in bacteria and cancer.
• Comprehensive functional insights into drug efflux transporters are limited.
• The bacterial ABC transporter EfrCD is a key target for studying efflux mechanisms.

Purpose of the Study:

• To systematically map the drug efflux activity of over 1,430 single variants of the EfrCD transporter.
• To elucidate the functional consequences of specific amino acid substitutions within the EfrCD transporter.
• To understand the transport mechanism and substrate binding site of EfrCD.

Main Methods:

• Deep mutational scanning (DMS) was employed to assess the efflux activity of EfrCD variants.
• Cryogenic electron microscopy (cryo-EM) was used to determine the structure of EfrCD.
• Analysis integrated mutational data with structural information of the inward-facing transporter.

Main Results:

• Introduction of negative charges in the substrate binding pocket enhanced efflux of positively charged ethidium.
• A high-affinity binding site and a peristaltic transport mechanism were identified.
• Specific mutations converted EfrCD into a drug importer, demonstrating functional plasticity.

Conclusions:

• Single mutations can significantly alter the function of ABC transporters like EfrCD.
• Understanding these mutations provides insights into drug resistance and transporter mechanisms.
• The study demonstrates the potential to engineer ABC transporters for specific import functions.