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

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-like motif in DrrB. This study lays a solid groundwork for examining roles of various conserved regions of DrrA and DrrB in transduction of conformational changes.

DnrI of Streptomyces peucetius binds to the resistance genes, drrAB and drrC but is activated by daunorubicin.

The master regulator, DnrI of Streptomyces peucetius is a member of the family of transcriptional activator, Streptomyces antibiotic regulatory proteins (SARP), which controls the biosynthesis of antitumor anthracycline, daunorubicin (DNR) and doxorubicin (DXR). The binding of DnrI to the heptameric repeat sequence found within the -35 promoter region of biosynthetic gene, dpsE activates it. To combat the increased level of intracellular DNR, the cell has developed self resistance mechanism mediated by drrAB and drrC genes which are regulated by regulatory genes. We find that a drug non-producing mutant, ΔdpsA, showed sensitive phenotype in plate assay along with an increased level of dnrI transcript. Whereas the mutant grown in the presence of DNR showed a resistant phenotype with a six and eight folds increase in drrAB and drrC transcripts respectively. Computational studies followed by molecular docking showed that DnrI bound as a monomer to a slightly modified heptameric DNA motif, 5'-ACACGCA in drrA and 5'-ACAACCT in drrC which was also proved by electrophoretic mobility shift assay. These findings confirm that DnrI belongs to winged helix-turn-helix DNA-binding protein with Tetratricopeptide Repeat domain. The transcriptional regulator DnrI binds to the resistance genes at specific sites but they are activated only when an increased load of intracellular DNR is sensed.

Characterization of a novel domain 'GATE' in the ABC protein DrrA and its role in drug efflux by the DrrAB complex.

A novel domain, GATE (Glycine-loop And Transducer Element), is identified in the ABC protein DrrA. This domain shows sequence and structural conservation among close homologs of DrrA as well as distantly-related ABC proteins. Among the highly conserved residues in this domain are three glycines, G215, G221 and G231, of which G215 was found to be critical for stable expression of the DrrAB complex. Other conserved residues, including E201, G221, K227 and G231, were found to be critical for the catalytic and transport functions of the DrrAB transporter. Structural analysis of both the previously published crystal structure of the DrrA homolog MalK and the modeled structure of DrrA showed that G215 makes close contacts with residues in and around the Walker A motif, suggesting that these interactions may be critical for maintaining the integrity of the ATP binding pocket as well as the complex. It is also shown that G215A or K227R mutation diminishes some of the atomic interactions essential for ATP catalysis and overall transport function. Therefore, based on both the biochemical and structural analyses, it is proposed that the GATE domain, located outside of the previously identified ATP binding and hydrolysis motifs, is an additional element involved in ATP catalysis.