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

Archives of Biochemistry and Biophysics
Sadia J Rahman, Parjit Kaur

Abstract

Bacterial multidrug transporter DrrAB exhibits overlapping substrate specificity with mammalian P-glycoprotein. DrrA hydrolyzes ATP, and the energy is transduced to carrier DrrB resulting in export of drugs. Previous studies suggested that DrrB contains a large and flexible drug-binding pocket made of aromatic residues contributed by several transmembrane helices with different drugs binding to both specific and shared residues in this pocket. However, direct binding of drugs to DrrAB or the mechanism of substrate-induced conformational changes between DrrA and DrrB has so far not been investigated. We used two fluorescence-based approaches to determine substrate binding to purified DrrAB. Our analysis shows that DrrB binds drugs with variable affinities and contains multiple drug binding sites. This work also provides evidence for two asymmetric nucleotide binding sites in DrrA with strikingly different binding affinities. Using targeted fluorescence labeling, we provide clear evidence of long-range conformational changes occurring between DrrA and DrrB. It is proposed that the transduction pathway from the nucleotide-binding DrrA subunit to the substrate binding DrrB subunit includes Q-loop and CREEM motifs in DrrA and EAA-li...Continue Reading

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Citations

Aug 16, 2019·The Journal of Biological Chemistry·Elizabeth PetersonParjit Kaur
Jun 30, 2019·Research in Microbiology·Cédric OrelleJean-Michel Jault
Mar 25, 2021·Chemical Reviews·Peter J F HendersonKarl A Hassan
Apr 11, 2021·Archives of Microbiology·Liuyang OuShuaiyin Chen

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