Photoaffinity labeling under non-energized conditions of a specific drug-binding site of the ABC multidrug transporter LmrA from Lactococcus lactis

Biochem Biophys Res Commun. 2003 Nov 21;311(3):696-701. doi: 10.1016/j.bbrc.2003.10.049.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / chemistry*
  • Antibiotics, Antineoplastic / pharmacology
  • Antineoplastic Agents, Phytogenic / pharmacology
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism
  • Binding Sites
  • Cell Membrane / metabolism
  • Colchicine / pharmacology
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Drug
  • Doxorubicin / pharmacology
  • Drug Resistance, Multiple
  • Ethidium / pharmacology
  • Fluorescent Dyes / pharmacology
  • Intercalating Agents / pharmacology
  • Lactococcus lactis / metabolism*
  • Light
  • Multidrug Resistance-Associated Proteins / chemistry*
  • Multidrug Resistance-Associated Proteins / metabolism
  • Peptides / chemistry
  • Precipitin Tests
  • Propionates / pharmacology
  • Protein Structure, Tertiary
  • Quinolines / pharmacology
  • Rhodamines / pharmacology
  • Vinblastine* / pharmacology

Substances

  • ATP-Binding Cassette Transporters
  • Antibiotics, Antineoplastic
  • Antineoplastic Agents, Phytogenic
  • Bacterial Proteins
  • DNA, Complementary
  • Fluorescent Dyes
  • Intercalating Agents
  • LmrA protein, Lactococcus lactis
  • Multidrug Resistance-Associated Proteins
  • Peptides
  • Propionates
  • Quinolines
  • Rhodamines
  • rhodamine 6G
  • verlukast
  • Vinblastine
  • Doxorubicin
  • Ethidium
  • Colchicine