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Construction of a Tryptophan‐less P‐glycoprotein by Directed Evolution for Mapping Drug Binding Sites
Author(s) -
Swartz Douglas,
Urbatsch Ina
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.714.14
Subject(s) - mutant , efflux , amino acid , atp binding cassette transporter , tryptophan , binding site , p glycoprotein , biochemistry , transporter , chemistry , wild type , mutagenesis , biology , multiple drug resistance , gene , antibiotics
P‐glycoprotein (Pgp) is an ATP Binding Cassette (ABC) transporter that functions as a multidrug efflux pump, causing drug resistance in cancer and other diseases. Polyspecific drug transport is likely achieved by drugs binding within sub‐pockets of a central cavity formed by the transmembrane domains (TMDs). However, little is known about the nature or location of drug binding in Pgp. Our objective is to use site‐specific tryptophan (Trp) fluorescence to map drug‐protein interactions. This has not been previously applied to Pgp due to 11 native Trps that contribute to a high intrinsic fluorescence. We used directed evolution to remove the native Trps while maintaining protein expression and function. Two successive rounds of site saturation mutagenesis were used to replace the 8 TMD Trps with the 19 other amino acids in Pgp. The mutants were subjected to a stringent selection process in Saccharomyces cerevisiae, using well established Pgp functional assays. Surviving mutants were sequenced to identify the amino acids that could successfully replace Trp at each site. This culminated in the identification of a mutant that lacks all 8 TMD Trps, and exhibits similar protein expression and activity as wild‐type Pgp. It also has significantly reduced the intrinsic Trp fluorescence signal, making it suitable for re‐introduction of Trps in the TMD to measure drug binding. Work is ongoing to remove the 3 remaining Trps from this mutant to produce a fully Trp‐less Pgp. We expect that this mutant will be a versatile new tool for studying Pgp using site‐specific Trp fluorescence.