Biochemical Characterization of Domains in the Membrane Subunit DrrB That Interact with the ABC Subunit DrrA:  Identification of a Conserved Motif

Abstract

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.

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

“…Primers were designed as described before (22). In the LDEVFL motif, conservative point mutations at positions 303 and 304 were created, which were named L303V and D304N, respectively.…”

“…Single Cysteine Substitutions in DrrA-pDx101 containing a single cysteine substitution in drrB at position 23 (S23C) was used as the template (22). Single cysteine substitution mutants were created at amino acid position 325, 323, 319, 311, 302, 287, 253, or 232 in DrrA in this clone.…”

“…Previous studies, using a cysteine to amine cross-linker, N-(gammamaleimidobutyryloxy)succinimide esters, have shown that the N-terminal cytoplasmic tail of DrrB (residues 1-53) is the region that contacts DrrA (22). To determine if the extreme C terminus of DrrA is the region that makes the above contact with the N-terminal tail of DrrB, cysteine substitutions were created in these two domains.…”

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).…”

“…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). Several homologues of DrrAB have been described for different species of drug-producing Streptomyces, conferring resistance to oleandomycin, tetronasin, mithramycin, and kasugamycin (151,220,279,404).…”