Conformational changes in a multidrug resistance ABC transporter DrrAB: Fluorescence-based approaches to study...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Bacterial multidrug transporter DrrAB shares substrate specificity with mammalian P-glycoprotein.
• Previous studies suggested a flexible drug-binding pocket in DrrB, but direct drug binding and conformational changes remained uninvestigated.

Purpose of the Study:

• To investigate direct drug binding to DrrAB.
• To elucidate the mechanism of substrate-induced conformational changes between DrrA and DrrB.

Main Methods:

• Utilized two fluorescence-based approaches to assess substrate binding to purified DrrAB.
• Employed targeted fluorescence labeling to detect conformational changes between DrrA and DrrB.

Main Results:

• Demonstrated that DrrB binds drugs with variable affinities at multiple sites.
• Identified two asymmetric nucleotide-binding sites in DrrA with distinct affinities.
• Provided evidence for long-range conformational changes between DrrA and DrrB.

Conclusions:

• The study reveals drug binding characteristics of DrrB and nucleotide binding of DrrA.
• Proposed a transduction pathway involving specific motifs (Q-loop, CREEM, EAA-like) for conformational changes.
• Establishes a foundation for further research into conserved regions of DrrA and DrrB in conformational transduction.