ABCMdb

ABCB3 p.Gly25Ala

Predicted by SNAP2:	A: D (63%), C: D (66%), D: D (80%), E: D (80%), F: N (61%), H: D (59%), I: D (66%), K: D (85%), L: N (61%), M: D (66%), N: D (63%), P: D (80%), Q: D (75%), R: D (80%), S: D (53%), T: D (63%), V: D (59%), W: D (75%), Y: N (61%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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[hide] Biochemical characterization of domains in the mem... Biochemistry. 2005 Feb 22;44(7):2661-70. Kaur P, Rao DK, Gandlur SM
Biochemical characterization of domains in the membrane subunit DrrB that interact with the ABC subunit DrrA: identification of a conserved motif.
Biochemistry. 2005 Feb 22;44(7):2661-70., 2005-02-22 [PMID:15709779]

Abstract [show]
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.

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253 These changes include S23A, G25A, E26D, and S35A in the N-terminal domain and C260A and A270S in the C-terminal domain. Login to comment
254 Doxorubicin resistance assays showed that S23A, G25A, and E26D mutations in the N-terminal domain confer sensitivity to between 4 and 6 µg/mL of doxorubicin while the cells containing the wild-type DrrA and DrrB grow up to 8-10 µg/mL of doxorubicin (Table 2). Login to comment
259 G25A and E26D also showed significantly reduced levels of DrrA and DrrB, although these are less affected as compared to S35I. Login to comment
267 Table 2: Doxorubicin Resistance of E. coli N43 Cells Expressing Wild-Type DrrA with DrrB Containing Mutations in the N-Terminal Cytoplasmic Tail or the C-Terminal Enda amt of dox, µg/mLdomain of DrrB location of cysteine 0 4 6 8 (wild type) C260 +++ +++ +++ ++ N-terminus S23A +++ ++ - - N-terminus G25A +++ ( - - N-terminus E26D +++ ( - - N-terminus S35A +++ +++ +++ +++ N-terminus S35I +++ ( - - C-terminus C260A +++ +++ +++ - C-terminus C260E +++ +++ +++ - C-terminus A270S +++ +++ +++ + C-terminus A270Y +++ +++ ++ ++ vector only pSU2718 +++ ( - - a Legend: +++, very good growth; ++ good growth; + some growth; -, no growth. Login to comment
327 Mutagenesis of certain residues in and around the conserved motif in the N terminus of DrrB, including S23A, G25A, E26D, and S35I, resulted in sensitivity to doxorubicin, although mutagenesis of C260 and A270 in the C terminus did not confer doxorubicin sensitivity significantly. Login to comment
331 Second-site suppressor analysis of the S23A, G25A, E26D, and S35I mutations will be carried out in the future to verify if these residues are indeed directly involved in interaction with DrrA, as is suggested by the cysteine cross-linking studies reported here. Login to comment
329 G25A, E26D, and S35I mutations also resulted in reduced levels of DrrA and DrrB. Login to comment