The Q-Loop of DrrA Is Involved in Producing the Closed Conformation of the Nucleotide Binding Domains and in Transduction of Conformational Changes between DrrA and DrrB

Abstract

DrrA and DrrB proteins form an ATP-dependent efflux pump for doxorubicin and daunorubicin in Streptomyces peucetius. DrrA, the catalytic subunit, forms a complex with the integral membrane protein DrrB. Previous studies have provided evidence for strong interaction between these two proteins, which was found to be critical for binding of ATP to DrrA and for stability of DrrB. Chemical cross-linking experiments carried out previously showed that in the resting state of the complex DrrA and DrrB are in contact with each other. Use of a cysteine-to-amine cross-linker then allowed identification of the N-terminal cytoplasmic tail of DrrB (residues 1-53) as the primary region of contact with DrrA. In this study, single-cysteine substitutions were introduced into different domains of DrrA in a strain already containing the S23C substitution in the N-terminal tail of DrrB. By using different arm-length disulfide cross-linkers, we found that a cysteine placed in the Q-loop region of DrrA traps DrrA in the dimeric state, thus indicating that in the closed conformation the Q-loops from opposing subunits are in the proximity of each other. Furthermore, the same region of DrrA was also found to interact with the N-terminus of DrrB, although the A-A interaction was much more prominent than the A-B interaction under these conditions. On the basis of additional data shown here, we propose that the interaction of the Q-loop with the N-terminal cytoplasmic tail of DrrB identifies an important step in the communication of conformational changes between DrrA and DrrB. The significance of these findings in the mechanism of the DrrAB complex is discussed, and a model based on analyses of different conformations of DrrA and DrrB is presented.

Functional Significance of the E Loop, a Novel Motif Conserved in the Lantibiotic Immunity ATP-Binding Cassette Transport Systems

“…the conformational transition of the Q loop and the coordination of this transition with the conformational changes of transmembrane subunits and NBDs (16). Similarly, it was suggested that the interaction of the Q loop in DrrA with the N-terminal cytoplasmic tail of DrrB, which is a transmembrane subunit, is involved in transmitting conformational changes between DrrA and DrrB (30). The X-ray structure of the fully assembled maltose transport system showed that the Q loops of MalK are in close proximity to the transmembrane subunit MalFG (24).…”

“…The key factor affecting the function of the NukFEG system needs to be determined. Recently, mutational studies of the Q loop have been carried out while studying DrrA, an NBD of an ABC transporter involved in self-resistance to anticancer antibiotics (doxorubicin and daunorubicin) (30). Nonconservative mutations of the Q loop (Q88S and Q88N) resulted in great reductions in the resistance levels; however, the Q88E mutation retained the resistance to a high degree.…”

The Extreme C Terminus of the ABC Protein DrrA Contains Unique Motifs Involved in Function and Assembly of the DrrAB Complex

“…Doxorubicin Resistance Assay-Doxorubicin resistance assays were carried out as described earlier (23). Briefly, the indicated plasmids were transformed into E. coli N43 cells, which are doxorubicin-sensitive.…”

“…ATP Bindng Assay-Photolabeling of DrrA with [␣-32 P]ATP was carried out in membranes containing wild type DrrAB or DrrAB bearing mutations in DrrA (23). The ATP binding assay was carried out in a 100-l reaction system containing buffer A, 0.1 mg of membrane protein, 10 M ATP (pH7.5), 10 Ci of [␣-32 P]ATP, 35 M doxorubicin, and 5 mM MgCl 2 .…”

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

“…Using gene fusions, a topology model has been proposed for DrrB, consisting of eight membrane-spanning segments with both N and C termini in the cytoplasm (164). Cysteine cross-linking revealed a motif within the N-terminal tail of DrrB that may be a modified version of the EAA motif present in ABC importers (236,392). These two components are biochemically coupled and are thought to form an efflux pump (236,237).…”