Photoaffinity labeling under non-energized conditions of a specific drug-binding site of the ABC multidrug transporter LmrA from Lactococcus lactis.
The Lactococcus lactis multidrug resistance ABC transporter protein LmrA has been shown to confer resistance to structurally and functionally diverse antibiotics and anti-cancer drugs. Using a previously characterized photoreactive drug analogue of Rhodamine 123 (iodo-aryl azido-Rhodamine 123 or IAARh123), direct and specific photoaffinity labeling of LmrA in enriched membrane vesicles could be achieved under non-energized conditions. This photoaffinity labeling of LmrA occurs at a physiologically relevant site as it was inhibited by molar excess of ethidium bromide>Rhodamine 6G>vinblastine>doxorubicin>MK571 (a quinoline-based drug) while colchicine had no effect. The MDR-reversing agents PSC 833 and cyclosporin A were similarly effective in inhibiting IAARh123 photolabeling of LmrA and P-glycoprotein. In-gel digestion with Staphyloccocus aureus V8 protease of IAARh123-photolabeled LmrA revealed several IAARh123 labeled polypeptides, in addition to a 6.8kDa polypeptide that comprises the last two transmembrane domains of LmrA.
Alqawi
Omar
O
Institute of Parasitology, McGill University, Macdonald Campus, Ste-Anne de Bellevue, Que, Canada.
Poelarends
Gerrit
G
Konings
Wil N
WN
Georges
Elias
E
Alqwai
Omar
O
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003078:Colchicine / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D004996:Ethidium / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D007364:Intercalating Agents / Q000494:pharmacology; D013294:Lactococcus lactis / Q000378:metabolism*; D008027:Light; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D011422:Propionates / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011804:Quinolines / Q000494:pharmacology; D012235:Rhodamines / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D000903:Antibiotics, Antineoplastic; D000972:Antineoplastic Agents, Phytogenic; D001426:Bacterial Proteins; D003078:Colchicine; D018076:DNA, Complementary; D004317:Doxorubicin; D004996:Ethidium; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010455:Peptides; D011422:Propionates; D011804:Quinolines; D012235:Rhodamines; D014747:Vinblastine; C101824:LmrA protein, Lactococcus lactis; C026188:rhodamine 6G; C059141:verlukast