Abstract
Pgp (P-glycoprotein) is a prototype ABC (ATP-binding-cassette) transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic Cys (cysteine) residues in Pgp with all 20 standard amino acids and selected for active mutants. From a pool of 75000 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 Cys427 (24 and 20%, respectively) and Cys1070 (37 and 25%) of the Walker A motifs in the NBDs (nucleotide-binding domains), Cys1223 in NBD2 (25 and 8%) and Cys638 in the linker region (24 and 16%), whereas close-by Cys669 tolerated glycine (16%) and alanine (14%), but not serine (absent). Cys1121 in NBD2 showed a clear preference for positively charged arginine (38%) suggesting a salt bridge with Glu269 in the ICL2 (intracellular loop 2) may stabilize domain interactions. In contrast, three Cys residues in transmembrane α-helices could be successfully replaced by alanine. The resulting CL (Cys-less) Pgp was fully active in yeast cells, and purified proteins displayed drug-stimulated ATPase activities indistinguishable from WT (wild-type) Pgp. Overall, directed evolution identified site-specific, non-conservative Cys substitutions that allowed building of a robust CL Pgp, an invaluable new tool for future functional and structural studies, and that may guide the construction of other CL proteins where alanine and serine have proven unsuccessful.
Highlights
Pgp (P-glycoprotein), known as ABCB1 or MDR1, is a member of the ABC (ATP-binding-cassette) transporter superfamily that functions as a multidrug efflux pump [1,2]
Polyspecific binding of transported drugs and Pgp inhibitors occurs in a central cavity formed by two transmembrane domain (TMD), but the precise locations and nature of these binding sites remain poorly defined [5,6]
Substitution of intracellular Cys residues Each of the nine native Cys residues in mouse Pgp is highly conserved among Pgp orthologues except Cys638 and Cys669, both located in the flexible linker that connects the two homologous halves of the protein (Supplementary Table S2 at http://www.bioscirep.org/bsr/034/bsr034e116add.htm)
Summary
Pgp (P-glycoprotein), known as ABCB1 or MDR1, is a member of the ABC (ATP-binding-cassette) transporter superfamily that functions as a multidrug efflux pump [1,2]. Mouse Pgp contains nine native Cys residues (2 more than human Pgp), with three located in the TMDs, three in the N-terminal NBD/linker region, and three in the C-terminal NBD (Figure 1).
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