Publication Date:
2014-05-15
Description:
P-glycoprotein (Pgp) is a prototype ATP binding cassette transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic cysteine (Cys) residues in Pgp with all twenty standard amino acids and selected for active mutants. From a pool of 75,000 transformants for each block of three Cys, we identified multiple mutants that preserved drug resistance and yeast mating activity. The most frequent substitutions were glycine and serine for C427 (24% and 20%, respectively) and C1070 (37% and 25%) of the Walker A motifs in the nucleotide binding domains (NBDs), C1223 in NBD2 (25% and 8%), and C638 in the linker region (24% and 16%), whereas close-by C669 tolerated glycine (16%) and alanine (14%), but not serine (absent). C1121 in NBD2 showed a clear preference for positively charged arginine (38%) suggesting a salt bridge with E269 in the intracellular loop ICL2 may stabilize domain interactions. In contrast, three Cys residues in transmembrane α-helices could be successfully replaced by alanine. The resulting Cys-less Pgp was fully active in yeast cells, and purified proteins displayed drug-stimulated ATPase activities indistinguishable from wild-type Pgp. Overall, directed evolution identified site-specific, non-conservative Cys substitutions that allowed building of a robust Cys-less Pgp, an invaluable new tool for future functional and structural studies, and that may guide the construction of other cysteine-less proteins where alanine and serine have proven unsuccessful.
Print ISSN:
0144-8463
Electronic ISSN:
1573-4935
Topics:
Biology
,
Chemistry and Pharmacology
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