Biochemical characterization of domains in the membrane subunit DrrB that interact with the ABC subunit DrrA: identification of a conserved motif.
DrrA and DrrB proteins confer resistance to the commonly used anticancer agents daunorubicin and doxorubicin in the producer organism Streptomyces peucetius. The drrAB locus has previously been cloned in Escherichia coli, and the proteins have been found to be functional in this host. DrrA, a soluble protein, belongs to the ABC family of proteins. It forms a complex with the integral membrane protein DrrB. Previous studies suggest that the function and stability of DrrA and DrrB are biochemically coupled. Thus, DrrA binds ATP only when it is in a complex with DrrB in the membrane. Further, DrrB is completely degraded if DrrA is absent. In the present study, we have characterized domains in DrrB that may be directly involved in interaction with DrrA. Several single-cysteine substitutions in DrrB were made. Interaction between DrrA and DrrB was studied by using a cysteine to amine chemical cross-linker that specifically cross-links a free sulfhydryl group in one protein (DrrB) to an amine in another (DrrA). We show here that DrrA cross-links with both the N- and the C-terminal ends of the DrrB protein, implying that they may be involved in interaction. Furthermore, this study identifies a motif within the N-terminal cytoplasmic tail of DrrB, which is similar to a motif recently shown by crystal structure analysis in BtuC and previously shown by sequence analysis to be also present in exporters, including MDR1. We propose that the motif present in DrrB and other exporters is actually a modified version of the EAA motif, which was originally believed to be present only in the importers of the ABC family. The present work is the first report where domains of interaction in the membrane component of an ABC drug exporter have been biochemically characterized.
Kaur
Parjit
P
Department of Biology, Georgia State University, Atlanta, Georgia 30303, USA.
Rao
Divya K
DK
Gandlur
Suvarna M
SM
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D017124:Conserved Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000235:genetics; D004317:Doxorubicin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D004352:Drug Resistance, Microbial; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D017384:Sequence Deletion / Q000235:genetics; D013388:Succinimides / Q000737:chemistry
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D003545:Cysteine; D004317:Doxorubicin; D008565:Membrane Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D021122:Protein Subunits; D013388:Succinimides; C104037:DrrA protein, Streptomyces peucetius; C104038:DrrB protein, Streptomyces peucetius; C032138:N-(gamma-maleimidobutyryloxy)succinimide