P-Glycoprotein Substrate Binding Domains Are Located at the Transmembrane Domain/Transmembrane Domain Interfaces: A Combined Photoaffinity Labeling-Protein Homology Modeling Approach

Abstract

P-glycoprotein (P-gp) is an energy-dependent multidrug efflux pump conferring resistance to cancer chemotherapy. Characterization of the mechanism of drug transport at a molecular level represents an important prerequisite for the design of pump inhibitors, which resensitize cancer cells to standard chemotherapy. In addition, P-glycoprotein plays an important role for early absorption, distribution, metabolism, excretion, and toxicity profiling in drug development. A set of propafenonetype substrate photoaffinity ligands has been used in this study in conjunction with matrix-assisted laser desorption/ionization timeof-flight mass spectrometry to define the substrate binding domain(s) of P-gp in more detail. The highest labeling was observed in transmembrane segments 3, 5, 8, and 11. A homology model for P-gp was generated on the basis of the dimeric crystal structure of Vibrio cholerae MsbA, an essential lipid transporter. Thereafter, the labeling pattern was projected onto the 3D atomic-detail model of P-gp to allow a visualization of the binding domain(s). Labeling is predicted by the model to occur at the two transmembrane domain/transmembrane domain interfaces formed between the amino-and carboxyl-terminal half of P-gp. These interfaces are formed by transmembrane (TM) segments 3 and 11 on one hand and TM segments 5 and 8 on the other hand. Available data on LmrA and AcrB, two bacterial multidrug efflux pumps, suggest that binding at domain interfaces may be a general feature of polyspecific drug efflux pumps.Multidrug resistance represents a serious obstacle to successful cancer chemotherapy. Although multifactorial in etiology, one type of multidrug resistance is associated with the overexpression of energy-dependent membrane-bound pumps, which intercept and efflux drugs before they reach their intracellular target structures. P-glycoprotein (ABCB1) represents a paradigm ATP-dependent efflux pump expressed in human cancer cells. In addition to its expression in cancer cells, P-gp is also physiologically expressed in a number of tissues such as intestinal epithelial cells, at the brush border of renal tubule epithelial cells, the canalicular side of hepatocytes, and in capillary endothelial cells forming the blood-brain barrier. It thus interferes with oral drug absorption and drug delivery to the brain, and it enhances renal and biliary excretion. P-gp has therefore attracted considerable attention as a nontarget in the field of drug development, because for a large number of active compounds, interaction with P-glycoprotein might compromise their future development into a drug. Considerable energy has therefore been devoted to the characterization of molecular features that make compounds P-gp substrates and to the definition of the molecular mechanism of drug transport by P-gp. A number of studies have dealt with the kinetics and thermodynamics of the transport process

Location of contact residues in pharmacologically distinct drug binding sites on P-glycoprotein

“…Supporting evidence is provided by the highly hydrophobic probe [ 125 I]-INA, which could only be covalently attached to P-gp in the presence of the substrate doxorubicin [38]. Location of a drug binding site proximal to the lipid-protein interface of P-gp was also provided by an elegant photoaffinity based approach [30]. P-gp was photolabelled by the substrate propafenone and then extensively digested.…”

“…The data involved mutagenesis [12][13][14][15], structural information [19], cysteine-directed mutagenesis [21,28,29] and mass spectroscopy [30]. The generation of recombinant baculovirus that produced expression of the P-gp isoforms was successful for seven single cysteine mutants and the cysteine-less (CL-P-gp) control ( Figure 1).…”

Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics

“…Site-directed mutagenesis of the full-length integrase followed by in vitro inhibition assays implicate two specific residues, C130 and W132, as being involved in inhibitor binding. The approach we used here is particularly useful for characterizing the stoichiometry of protein-ligand complexes that are difficult to analyze by X-ray crystallography and NMR techniques.With regard to drug development and design, once information regarding the location and stoichiometry of protein-ligand interactions is obtained, the next step is to develop 3D models of the complex [32,38,46]. Structural models of protein-binding sites can then be used to conduct molecular modeling experiments to identify novel compounds that may have high affinity for the binding site, or screen the affinity of existing compounds computationally.…”

“…With regard to drug development and design, once information regarding the location and stoichiometry of protein-ligand interactions is obtained, the next step is to develop 3D models of the complex [32,38,46]. Structural models of protein-binding sites can then be used to conduct molecular modeling experiments to identify novel compounds that may have high affinity for the binding site, or screen the affinity of existing compounds computationally.…”

“…Subsequent proteolytic digestion of the isolated protein-ligand complex and MS-based sequencing can, in turn, reveal the sequence of the amino acids in the ligand-binding region and provide valuable information regarding the potential binding chemistry. These techniques have been used to define a wide variety of binding sites on protein receptors including those for allosteric regulation of enzyme substrate specificity [29], bile acid transport protein [30], alkyl glucoside inhibitors of glucose cotransport protein [31], allosteric inhibitors of protein-protein interactions [32], inhibitors of enzyme activity [33], visual cycle enzyme substrates [34], substrate-binding sites of cytochrome P450 [35], and substrate-binding domains of the drug-efflux mediator P-glycoprotein [36][37][38]. In addition to identifying and characterizing binding sites in known protein receptors, several groups have demonstrated the promise of combining proteomics with PAL to identify protein targets for candidate drugs [39,40].…”