Global alteration of the drug-binding pocket of human P-glycoprotein (ABCB1) by substitution of fifteen conserved residues reveals a negative correlation between substrate size and transport efficiency

Abstract

P-glycoprotein (P-gp), an ATP-dependent efflux pump, is linked to the development of multidrug resistance in cancer cells. However, the drug-binding sites and translocation pathways of this transporter are not yet well-characterized. We recently demonstrated the important role of tyrosine residues in regulating P-gp ATP hydrolysis via hydrogen bond formations with high affinity modulators. Since tyrosine is both a hydrogen bond donor and acceptor, and non-covalent interactions are key in drug transport, in this study we investigated the global effect of enrichment of tyrosine residues in the drug-binding pocket on the drug binding and transport of P-gp. By employing computational analysis, 15 conserved residues in the drug-binding pocket of human P-gp that interact with substrates were identified and then substituted with tyrosine, including 11 phenylalanine (F72, F303, F314, F336, F732, F759, F770, F938, F942, F983, F994), two leucine (L339, L975), one isoleucine (I306), and one methionine (M949). Characterization of the tyrosine-rich P-gp mutant in HeLa cells demonstrated that this major alteration in the drug-binding pocket by introducing fifteen additional tyrosine residues is well tolerated and has no measurable effect on total or cell surface expression of this mutant. Although the tyrosine-enriched mutant P-gp could transport small to moderate size (<1000 Daltons) fluorescent substrates, its ability to transport large (>1000 Daltons) substrates such as NBD-cyclosporine A, Bodipy-paclitaxel and Bodipy-vinblastine was significantly decreased. This was further supported by the physico-chemical characterization of seventeen tested substrates, which revealed a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant.

Evidence for the critical role of transmembrane helices 1 and 7 in substrate transport by human P-glycoprotein (ABCB1)

“…In the drug-binding pocket of P-gp, there are at least forty residues known to interact with substrates, based on earlier mutagenesis studies, modeling and co-crystallization data of mouse P-gp [ 16 , 24 – 27 , 32 , 33 , 45 – 49 ]. For our mutagenesis studies, we selected six residues each in TMH1 and TMH7 that are homologous to each other in sequence when aligned.…”

“…HeLa cells were transduced with the WT or TMH1,7 mutant P-gp BacMam baculovirus at a titer of 30–100 virus particles per cell, as described previously [ 16 , 17 , 35 ]. Twenty-two hours post-transduction, the cells were trypsinized and harvested.…”

“…A number of studies have shown the existence of overlapping binding sites for different substrates as well as multiple binding sites for a given substrate, indicating the involvement of multiple residues within the drug-binding pocket [ 17 , 23 , 30 – 33 ]. In a recent study, we generated a mutant of P-gp termed 15Y with fifteen conserved residues mutated to tyrosine to determine the extent of flexibility of the drug-binding pocket [ 16 ]. The 15Y mutant was stably expressed and was able to transport most of the small and medium-size substrates tested, although the substrates with large molecular weight (>1000 Da) were not transported, demonstrating the extent of flexibility of the drug-binding pocket of human P-gp.…”

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

“…Complex competitive and mixed-type inhibition were also observed in drug-stimulated ATPase assays suggesting positional overlap of classical transport substrates with ligands localized in the structures 39 . The structures have motivated a flurry of site directed mutagenesis studies to probe contact residues for binding of cancer drugs and classical substrates 32,[40][41][42] (reviewed in 3 ) as well as other inhibitors for which structures are not yet available, supported by molecular dynamics and docking simulations [43][44][45][46] . The emerging picture is that of spatially distinct binding sites that can simultaneously bind multiple drugs, as well as spatially overlapping binding sites for certain drugs that are linked through cooperative and non-cooperative interactions 3,41,42,45 .…”

ABC transporters confer multidrug resistance to Drosophila intestinal stem cells

“…The other possibility, not mutually exclusive with the first, is that the stem cells have intrinsic multidrug resistance properties, as described by the efflux properties of vertebrate stem cells 32 . To examine both possibilities further, we fed flies a library of 10 red fluorescent dyes, nine of which are membrane permeable and are used as indicators of drug efflux 33,34 . These include the rhodamine derivatives Tetramethylrhodamine (TMRM) and Rhodamine 123 as well as seven Syto Red dyes that range in size from 450 to 550 MW.…”