Simultaneous Binding of Two Different Drugs in the Binding Pocket of the Human Multidrug Resistance P-glycoprotein

Loo, Tip W.; Bartlett, M. Claire; Clarke, David M.

doi:10.1074/jbc.m308559200

Cited by 177 publications

(160 citation statements)

References 49 publications

(37 reference statements)

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“…Each TMD contains six TM segments that can interact with each other when expressed as separate polypeptides or when the NBDs are deleted (35). Cysteine-scanning mutagenesis of the TM segments and reaction of the cysteine mutants with thiol-reactive drug substrates indicate that TM4 -6 in the Nterminal half and TM9 -12 in the C-terminal half contribute residues to the common drug-binding pocket (25,29,40). This pocket is shaped like a funnel, with the narrowest part at the cytoplasmic ends of the TM segments (27).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Val133 and Cys137 in Transmembrane Segment 2 Are Close to Arg935 and Gly939 in Transmembrane Segment 11 of Human P-glycoprotein

Loo

Bartlett

Clarke

2004

Journal of Biological Chemistry

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The human multidrug resistance P-glycoprotein (P-gp 1 ; ABCB1) uses ATP to transport a wide variety of structurally unrelated compounds of different sizes from the cell. The physiological function of P-gp is unknown. It is present in relatively higher levels in some organs such as the intestine, kidney, and blood-brain/testes barrier and therefore may function to protect the organism from toxins in the diet and the environment (1-3). Its relatively high expression in these organs can affect the therapeutic efficacy of oral drugs, whereas overexpression of P-gp in some tumor cells can complicate cancer chemotherapy regimens (4, 5).P-gp is a member of the ATP-binding cassette family of transporters (6). Its 1280 amino acids are arranged as two homologous halves with 43% amino acid identity. A linker region of ϳ60 amino acids connects the two halves of the protein (7). Each half has six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site (8, 9). The protein functions as a monomer (10). Each half of P-gp has basal ATPase activity, but drug-stimulated ATPase activity or conferral of drug resistance requires the presence of both halves of the protein (11, 12). Both halves do not have to be covalently linked for function (11). Both nucleotide-binding domains (NBDs) are essential for activity (13)(14)(15), and the two ATP molecules likely interact at the interface of the Walker A site in one NBD and the LSGGQ consensus site in the other NBD (16). Drug substrates that stimulate or inhibit ATPase activity cause these sequences to come closer or to move farther apart, respectively (17).The TM segments likely interact with drug substrates in the lipid bilayer (18 -20). Studies on P-gp deletion mutants show that the transmembrane domains (TMDs) alone can bind drug substrates (12). Drug substrates bind at distinct regions in a common drug-binding pocket that is formed by the interface between the TMDs of both halves of P-gp (21-25). Disulfide cross-linking studies and labeling of cysteine mutants with thiol-reactive drug substrates indicate that the two halves are arranged in a "head-to-tail" arrangement (26), with TM4 -6 of TMD1 (the N-terminal TMD containing TM1-6) and TM9 -12 of TMD2 (the C-terminal TMD containing TM7-12) forming the drug-binding pocket (25,27). Drug binding involves an induced fit mechanism (28), and only binding in some orientations can allow for conformational changes to induce ATP hydrolysis (29,30). Therefore, knowledge about the packing of the TM segments between the two halves of P-gp is important for understanding the mechanism of P-gp.The drug-binding pocket is "funnel-shaped," narrow at the cytoplasmic side and wider at the extracellular end (27). We recently showed that the homologous halves of P-gp are in close contact at the cytoplasmic ends of TM5 and TM8 (31). We predicted the presence of another contact point between TM2 or TM3 with TM11 in the two halves of P-gp for the formation of the funnel-shaped drug-binding pocket. In this study, we used cysteine-scann...

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Section: Resultsmentioning

confidence: 99%

“…Perhaps binding of rhodamine B caused TM2 and TM11 to move apart. Alternatively, binding of rhodamine B may have caused at least one of the TM segments to rotate in P-gp (28,40,55). Other drug substrates such as Hoechst 33342 and vinblastine inhibited cross-linking of only mutant V133C(TM2)/G939C(TM11).…”

Section: Discussionmentioning

confidence: 99%

Val133 and Cys137 in Transmembrane Segment 2 Are Close to Arg935 and Gly939 in Transmembrane Segment 11 of Human P-glycoprotein

Loo

Bartlett

Clarke

2004

Journal of Biological Chemistry

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“…Mdr transporters from various families were shown to contain multiple drug interaction sites, and these sites are often allosterically linked (18,19). In MdfA, binding of Cm was shown to increase the binding affinity for TPP (9).…”

Section: Discussionmentioning

confidence: 99%

A Promiscuous Conformational Switch in the Secondary Multidrug Transporter MdfA

Fluman

Cohen-Karni

Weiss

et al. 2009

Journal of Biological Chemistry

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Multidrug (Mdr) transporters are membrane proteins that actively export structurally dissimilar drugs from the cell, thereby rendering the cell resistant to toxic compounds. Similar to substrate-specific transporters, Mdr transporters also undergo substrate-induced conformational changes. However, the mechanism by which a variety of dissimilar substrates are able to induce similar transport-compatible conformational responses in a single transporter remains unclear. To address this major aspect of Mdr transport, we studied the conformational behavior of the Escherichia coli Mdr transporter MdfA. Our results show that indeed, different substrates induce similar conformational changes in the transporter. Intriguingly, in addition, we observed that compounds other than substrates are able to confer similar conformational changes when covalently attached at the putative Mdr recognition pocket of MdfA. Taken together, the results suggest that the Mdr-binding pocket of MdfA is conformationally sensitive. We speculate that the same conformational switch that usually drives active transport is triggered promiscuously by merely occupying the Mdr-binding site.

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“…There are 2 portals (formed by TMs 4/6 and 10/12) that allow hydrophobic molecules to enter the cavity directly from the inner leaflet. The drug binding pocket generally contains hydrophobic and aromatic residues and is large enough to accommodate at least two substrate molecules simultaneously [25] . In fact, this was supported by the X-ray structure reported by Aller et al [24] .…”

Section: Introductionmentioning

confidence: 99%

How to overcome ATP-binding cassette drug efflux transporter-mediated drug resistance?

Jaramillo¹,

Saig²,

Cloos³

et al. 2018

CDR

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P-glycoprotein (ABCB1), multidrug resistance protein-1 (ABCC1) and breast cancer resistance protein (ABCG2) belong to the ATP-binding cassette (ABC) superfamily of proteins that play an important physiological role in protection of the body from toxic xenobiotics and endogenous metabolites. Beyond this, these transporters determine the toxicity profile of many drugs, and confer multidrug resistance (MDR) in cancer cells associated with a poor treatment outcome of cancer patients.It has long been hypothesized that inhibition of ABC drug efflux transporters will increase drug accumulation and thereby overcome MDR, but until now no approved inhibitor of these transporters is available in the clinic. In this review we present molecular strategies to overcome this type of drug resistance and discuss for each of these strategies their promising value or indicate underlying reasons for their limited success.

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Simultaneous Binding of Two Different Drugs in the Binding Pocket of the Human Multidrug Resistance P-glycoprotein

Cited by 177 publications

References 49 publications

Val133 and Cys137 in Transmembrane Segment 2 Are Close to Arg935 and Gly939 in Transmembrane Segment 11 of Human P-glycoprotein

Val133 and Cys137 in Transmembrane Segment 2 Are Close to Arg935 and Gly939 in Transmembrane Segment 11 of Human P-glycoprotein

A Promiscuous Conformational Switch in the Secondary Multidrug Transporter MdfA

How to overcome ATP-binding cassette drug efflux transporter-mediated drug resistance?

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