P-glycoprotein is fully active after multiple tryptophan substitutions

Abstract

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.

Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance

“…This selection scheme is based on the ability to convey resistance of the yeast to fungicides as well as export a-factor pheromone and restore mating in the otherwise sterile yeast by complementing for Ste6, a Pgp yeast homologue [34,39]. Mating, by itself, is a stringent test of mutant function as we previously demonstrated [40]. The customized selection scheme was designed to identify mutants that could transport multiple, structurally diverse substrates and preserve polyspecificity, an important quality of Pgp.…”

About P‐glycoprotein: a new drugable domain is emerging from structural data

“…Finally, the authors demonstrated that the F1086A mutation disrupted the NBD/TMD coupling because F1086A successfully inhibited ATP‐dependent cross‐linking of the mutant protein T333C/L975C, located at the extracellular ends of TM helices 6 and 12 . Another study also pointed out the importance of TMD/NBD interdomain communication by showing that mutations in tryptophans located at the coupling helices (W158F/W799F) severely impaired mating efficiencies in yeast colonies (Table ) …”

“…122 Another study also pointed out the importance of TMD/NBD interdomain communication by showing that mutations in tryptophans located at the coupling helices (W158F/ W799F) severely impaired mating efficiencies in yeast colonies ( Table 2). 132 Two other mutations in ICH1 (D164C) and ICH3 (D805C) were also described to have significant alterations in P-gp function. While D805C completely abolished drug transport, the double mutant D164C/D805C showed about 70% decrease in daunorubicin and [N-ε(4-nitrobenzofurazan-7-yl)-d-Lys 8 ]-cyclosporine A (NBD-CsA) transport, only partially effluxing calcein-AM (~60%).…”

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions

“…Positive clones were then screened in two different fungicidal drugs, FK506 and doxorubicin. This selection scheme was designed to identify mutants that could preserve polyspecific drug transport, an important quality of Pgp, based on its ability to export the a-mating factor and convey fungicidal resistance to yeast against two drugs 74,78,79 . The blocks from clones that mated and were active in both drug assays were sequenced to identify Trp-free clones.…”

“…The first attempt to create Trp-free Pgp by replacing all 11 Trps by Phe ("All-F") 73 gave a transporter with minimal function and impaired expression, making it unusable as a tool for further studies. Subsequent experiments showed that multiple Pgp Trps could be successfully removed, but also suggested that aromatic amino acids may not be the ideal substitute at several Pgp Trp locations 74 . In the context of a large integral membrane protein, Trps can be found in a variety of local environments, as demonstrated by Pgp.…”