P-glycoprotein is fully active after multiple tryptophan substitutions
| Autor: | Ina L. Urbatsch, Douglas J. Swartz, Joachim Weber |
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| Rok vydání: | 2013 |
| Předmět: |
Models
Molecular Drug binding sites Membrane bilayer Antifungal Agents Saccharomyces cerevisiae Mutant Molecular Conformation Biophysics Plasma protein binding P-glycoprotein Biology Crystallography X-Ray Biochemistry Article Tacrolimus Cell membrane Drug Resistance Fungal medicine Humans ATP Binding Cassette Transporter Subfamily B Member 1 Binding site Adenosine Triphosphatases Binding Sites Valinomycin Dose-Response Relationship Drug Cell Membrane Tryptophan Conservative tryptophan substitutions Polyspecificity Cell Biology biology.organism_classification Native tryptophans medicine.anatomical_structure Verapamil Mutation Cyclosporine biology.protein Intracellular Protein Binding |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Biomembranes. 1828:1159-1168 |
| ISSN: | 0005-2736 |
| DOI: | 10.1016/j.bbamem.2012.12.005 |
| Popis: | P-glycoprotein (Pgp) is an important contributor to multidrug resistance of cancer. Pgp contains eleven native tryptophans (Trps) that are highly conserved among orthologs. We replaced each Trp by a conservative substitution to determine which Trps are important for function. Individual Trp mutants W44R, W208Y, W132Y, W704Y and W851Y, situated at the membrane surface, revealed significantly reduced Pgp induced drug resistance against one or more fungicides and/or reduced mating efficiencies in Saccharomyces cerevisiae. W158F and W799F, located in the intracellular coupling helices, abolished mating but retained resistance against most drugs. In contrast, W228F and W311Y, located within the membrane, W694L, at the cytoplasmic membrane interface, and W1104Y in NBD2 retained high levels of drug resistance and mating efficiencies similar to wild-type Pgp. Those were combined into pair (W228F/W311Y and W694L/W1104Y) and quadruple (W228F/W311Y/W694L/W1104Y) mutants that were fully active in yeast, and could be purified to homogeneity. Purified pair and quad mutants exhibited drug-stimulated ATPase activity with binding affinities very similar to wild-type Pgp. The combined mutations reduced Trp fluorescence by 35%, but drug induced fluorescence quenching was unchanged from wild-type Pgp suggesting that several membrane-bound Trps are sensitive to drug binding. Overall, we conclude that Trps at the membrane surface are critical for maintaining the integrity of the drug binding sites, while Trps in the coupling helices are important for proper interdomain communication. We also demonstrate that functional single Trp mutants can be combined to form a fully active Pgp that maintains drug polyspecificity, while significantly reducing intrinsic fluorescence. |
| Databáze: | OpenAIRE |
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