Membrane Topology of the DrrB Protein of the Doxorubicin Transporter of Streptomyces peucetius

Abstract

Daunorubicin and doxorubicin, two commonly used anticancer agents, are produced by the soil bacterium Streptomyces peucetius. Self-resistance to these antibiotics in S. peucetius is conferred by the drrAB locus that codes for two proteins, DrrA and DrrB. DrrA is an ATPbinding protein. It belongs to the ABC family of transporters and shares sequence and functional similarities with P-glycoprotein of cancer cells. DrrB is an integral membrane protein that might function as a transporter for the efflux of daunorubicin and doxorubicin. Together, DrrA and DrrB are believed to form an ATPdriven pump for the efflux of these drugs. The drrAB locus has been cloned, and the two proteins have been expressed in a functional form in Escherichia coli. A topological analysis of the DrrB protein was performed using gene fusion methodology. Random and site-directed fusions of the drrB gene to lacZ, phoA, or gfp reporter genes were created. Based on the fusion data, a topological model of the DrrB protein is proposed in which the protein has eight membrane-spanning domains with both the N terminus and the C terminus in the cytoplasm.

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

“…DrrA consists of an ABC-type NBD, while DrrB is an IM protein. 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).…”

The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse

“…In addition, RHA1 likely produces several bioactive compounds of its own (see below). Indeed, DrrA (ro06841) and DrrB (ro06840) homologs share 65% and 51% amino acid sequence identity with the daunorubicin transporters from Streptomyces peucetius (16). Although many MFS transporters in RHA1 are homologs of tetracycline transporters (e.g., ro04399), RHA1 is not resistant to tetracycline, suggesting that these proteins transport other compounds.…”

Dual Role of the Metalloprotease FtsH in Biogenesis of the DrrAB Drug Transporter

“…However, in the absence of functional FtsH, the DrrB protein accumulates even in the absence of DrrA confirming that FtsH monitors the folding status of DrrB and removes it if it is improperly assembled. The molecular details of proteolysis of DrrB by FtsH are currently unknown, however, based on the prevalent model for its action (8,26) we assume that FtsH could initiate proteolysis of DrrB either at the N-or the C-terminal end (both of which are found in the cytoplasm (27)). Crosslinking studies previously showed that the N terminus of DrrB is the major site of interaction with DrrA (3,27); therefore we propose that proteolysis of DrrB initiates at its N-terminal tail, and binding of DrrA to this region of DrrB protects it from proteolysis by FtsH.…”