The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform
Osama K Abouzied - One of the best experts on this subject based on the ideXlab platform.
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understanding the physical and chemical nature of the warfarin Drug Binding Site in human serum albumin experimental and theoretical studies
Current Pharmaceutical Design, 2015Co-Authors: Osama K AbouziedAbstract:Abstract Human serum albumin (HSA) is one of the major carrier proteins in the body and constitutes approximately half of the protein found in blood plasma. It plays an important role in lipid metabolism, and its ability to reversibly bind a large variety of pharmaceutical compounds makes it a crucial determinant of Drug pharmacokinetics and pharmacodynamics. This review deals with one of the protein's major Binding Sites "Sudlow I" which includes a Binding pocket for the Drug warfarin (WAR). The Binding nature of this important Site can be characterized by measuring the spectroscopic changes when a ligand is bound. Using several Drugs, including WAR, and other Drug-like molecules as ligands, the results emphasize the nature of Sudlow I as a flexible Binding Site, capable of Binding a variety of ligands by adapting its Binding pockets. The high affinity of the WAR pocket for Binding versatile molecular structures stems from the flexibility of the amino acids forming the pocket. The Binding Site is shown to have an ionization ability which is important to consider when using Drugs that are known to bind in Sudlow I. Several studies point to the important role of water molecules trapped inside the Binding Site in molecular recognition and ligand Binding. Water inside the protein's cavity is crucial in maintaining the balance between the hydrophobic and hydrophilic nature of the Binding Site. Upon the unfolding and refolding of HSA, more water molecules are trapped inside the Binding Site which cause some swelling that prevents a full recovery from the denatured state. Better understanding of the mechanism of Binding in macromolecules such as HSA and other proteins can be achieved by combining experimental and theoretical studies which produce significant synergies in studying complex biochemical phenomena.
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spectroscopy of hydroxyphenyl benzazoles in solution and human serum albumin detecting flexibility specificity and high affinity of the warfarin Drug Binding Site
RSC Advances, 2013Co-Authors: Osama K AbouziedAbstract:The complex photophysics of 2-(2′-hydroxyphenyl)benzoxazole (HBO), 2-(2′-hydroxyphenyl)benzimidazole (HBI), and 2-(2′-hydroxyphenyl)benzothiazole (HBT) in different media makes them suitable as fluorescent probes to study the nature of Binding Sites in macromolecular systems. In this work, we investigate the spectroscopy of the three benzazole derivatives (HBXs) in different solvents and in human serum albumin (HSA) in order to understand the Binding mechanism in subdomain IIA of HSA which has the ability to host a large variety of natural and pharmaceutical compounds. The three probes are found to specifically bind close to W214, the sole tryptophan residue in HSA, in a mode similar to that of the Binding of the anticoagulant Drug warfarin. The current results show that the structural differences between the three HBX molecules did not produce any measurable effects when Binding with HSA. In particular, the change in planarity of the molecular backbone, from a perfectly planar and more rigid structure (HBO) to a twisted structure (HBI) and a flexible structure (HBT) has no effect on the mode of Binding. Also, the strength of the intramolecular hydrogen bonds in HBXs (HBO > HBT > HBI) is shown not to intervene with the ability of HSA to ionize the ligands via through-space interaction with polar amino acid residues, similar to enzymatic reactions. The results emphasize the nature of HSA as a versatile and indiscriminate receptor, capable of Binding a variety of ligands by adapting its Binding pockets. In this regard, Binding of HBXs, and other structurally similar ligands, in subdomain IIA is best described by the induced-fit model in which considerable flexibility of the Binding Site is necessary for molecular recognition. The results also point to the high affinity of the warfarin Binding pocket (within subdomain IIA) for Binding versatile molecular structures including several Drugs. This affinity stems from the flexibility of the amino acids forming the Binding pocket.
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exploring the Drug Binding Site sudlow i of human serum albumin the role of water and trp214 in molecular recognition and ligand Binding
ChemPhysChem, 2011Co-Authors: Osama K Abouzied, Najla AllawatiaAbstract:Binding interactions that occur between proteins and low molecular weight molecules are the initiators of most of the biochemical reactions observed in vitro and in vivo. One example of such interactions is the Binding between the protein human serum albumin (HSA) and small molecules including several Drugs. HSA is the most abundant protein in plasma and constitutes approximately half of the protein found in human blood. This protein of 585 residues is composed of a single polypeptide chain, with three a-helical domains I-III, each containing two subdomains A and B (Figure 1). The pro-
Arpad Mike - One of the best experts on this subject based on the ideXlab platform.
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Different pH-sensitivity patterns of 30 sodium channel inhibitors suggest chemically different pools along the access pathway
Frontiers in Pharmacology, 2015Co-Authors: Alexandra Lazar, Krisztina Pesti, Nora Lenkey, Laszlo Fodor, Arpad MikeAbstract:The major Drug Binding Site of sodium channels is inaccessible from the extracellular side, Drug molecules can only access it either from the membrane phase, or from the intracellular aqueous phase. For this reason, ligand-membrane interactions are as important determinants of inhibitor properties, as ligand-protein interactions. One-way to probe this is to modify the pH of the extracellular fluid, which alters the ratio of charged vs. uncharged forms of some compounds, thereby changing their interaction with the membrane. In this electrophysiology study we used three different pH values: 6.0, 7.3, and 8.6 to test the significance of the protonation-deprotonation equilibrium in Drug access and affinity. We investigated Drugs of several different indications: carbamazepine, lamotrigine, phenytoin, lidocaine, bupivacaine, mexiletine, flecainide, ranolazine, riluzole, memantine, ritanserin, tolperisone, silperisone, ambroxol, haloperidol, chlorpromazine, clozapine, fluoxetine, sertraline, paroxetine, amitriptyline, imipramine, desipramine, maprotiline, nisoxetine, mianserin, mirtazapine, venlafaxine, nefazodone, and trazodone. We recorded the pH-dependence of potency, reversibility, as well as onset/offset kinetics. As expected, we observed a strong correlation between the acidic dissociation constant (pKa) of Drugs and the pH-dependence of their potency. Unexpectedly, however, the pH-dependence of reversibility or kinetics showed diverse patterns, not simple correlation. Our data are best explained by a model where Drug molecules can be trapped in at least two chemically different environments: A hydrophilic trap (which may be the aqueous cavity within the inner vestibule), which favors polar and less lipophilic compounds, and a lipophilic trap (which may be the membrane phase itself, and/or lipophilic Binding Sites on the channel). Rescue from the hydrophilic and lipophilic traps can be promoted by alkalic and acidic extracellular pH, respectively.
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the enigmatic Drug Binding Site for sodium channel inhibitors
Current Molecular Pharmacology, 2010Co-Authors: Arpad Mike, Peter LukacsAbstract:: Local anesthetics have been in clinical use since 1884, and different aspects of the local anesthetic Binding Site have been studied in enormous detail. In spite of all these efforts, some of the most fundamental questions--such as which exact residues constitute the Binding Site, how many Binding Sites exist, do local anesthetics share their Binding Site(s) with other sodium channel inhibitors, and what are the mechanisms of inhibition--are still largely unanswered. We review accumulated data on the "local anesthetic receptor"and discuss controversial points, such as possible mechanisms of inhibition, the possibility of additional Binding Sites, the orientation of S6 helices, and the internal vs. external position of the anticonvulsant Binding Site. We describe the four following specific groups of functionally important residues: i) conserved asparagines six residues below the hinge residues; we propose that they are oriented toward the external surface of S6 helices, and have a critical role in the coupling of voltage sensors to gating, ii) residues lining the inner vestibule and constructing the "orthodox" Binding Site, iii) residues around the outer vestibule, which have been proposed to constitute an alternative external Binding Site, and iv) residues determining external access for quaternary amine inhibitors, such as QX314. We conclude that sodium channel inhibitors must be heterogenous in terms of Binding Sites and inhibition mechanisms, and propose that this heterogeneity should be taken into consideration during Drug development.
David M Clarke - One of the best experts on this subject based on the ideXlab platform.
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Permanent activation of the human P-glycoprotein by covalent modification of a residue in the Drug-Binding Site.
Journal of Biological Chemistry, 2003Co-Authors: M. Claire Bartlett, David M ClarkeAbstract:Abstract The human multiDrug resistance P-glycoprotein (ABCB1) transports a broad range of structurally diverse compounds out of the cell. The transport cycle involves coupling of Drug Binding in the transmembrane domains with ATP hydrolysis. Compounds such as verapamil stimulate ATPase activity. We used cysteine-scanning mutagenesis of the transmembrane segments and reaction with the thiol-reactive substrate analog of verapamil, methanethiosulfonate (MTS)-verapamil, to test whether it caused permanent activation of ATP hydrolysis. Here we report that one mutant, I306C(TM5) showed increased ATPase activity (8-fold higher than untreated) when treated with MTS-verapamil and isolated by nickel-chelate chromatography. Drug substrates that either enhance (calcein acetoxymethyl ester, demecolcine, and vinblastine) or inhibit (cyclosporin A and trans-(E)-flupentixol) ATPase activity of Cys-less or untreated mutant I306C P-glycoprotein did not affect the activity of MTS-verapamil-treated mutant I306C. Addition of dithiothreitol released the covalently attached verapamil, and ATPase activity returned to basal levels. Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil, vinblastine, or colchicine prevented activation of mutant I306C by MTS-verapamil. The results suggest that MTS-verapamil reacts with I306C in a common Drug-Binding Site. Covalent modification of I306C affects the long range linkage between the Drug-Binding Site and the distal ATP-Binding Sites. This results in the permanent activation of ATP hydrolysis in the absence of transport. Trapping mutant I306C in a permanently activated state indicates that Ile-306 may be part of the signal to switch on ATP hydrolysis when the Drug-Binding Site is occupied.
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vanadate trapping of nucleotide at the atp Binding Sites of human multiDrug resistance p glycoprotein exposes different residues to the Drug Binding Site
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: David M ClarkeAbstract:The human multiDrug resistance P-glycoprotein uses ATP to transport a wide variety of structurally unrelated cytotoxic compounds out of the cell. In this study, we used cysteine-scanning mutagenesis and cross-linking studies to identify residues that are exposed to the Drug-Binding Site upon vanadate trapping. In the absence of nucleotides, C222(TM4) was cross-linked to C868(TM10) and C872(TM10); C306(TM5) was cross-linked to C868(TM10), C872(TM10), C945(TM11), C982(TM12), and C984(TM12); and C339(TM6) was cross-linked to C868(TM10), C872(TM10), C942(TM11), C982(TM12), and C985(TM12). These cysteines are in the middle of the predicted transmembrane (TM) segments and form the Drug-Binding Site. Cross-linking between 332C(TM6) and cysteines introduced at the extracellular side of other TM segments was also done. In the absence of nucleotides, residues 332C and 856C on the extracellular side of TMs 6 and 10, respectively, were cross-linked with a 13-A cross-linker (M8M, 3,6-dioxaoctane-1,8-diyl bismethanethiosulfonate). ATP plus vanadate inhibited cross-linking between 332C(TM6) and 856C(TM10) as well as those in the Drug-Binding Site. Instead, vanadate trapping promoted cross-linking between 332C(TM6) and 976C(TM12) with a 10-A cross-linker (M6M, 1,6-hexanediyl bismethanethiosulfonate). When ATP hydrolysis was allowed to proceed, then 332C(TM12) could form a disulfide bond with 975C(TM12). The cross-linking pattern of 332C(TM6) with residues in TM10 and TM12 indicates that the Drug-Binding Site undergoes dynamic and relatively large conformational changes, and that different residues are exposed to the Drug-Binding Site during the resting phase, upon vanadate trapping and at the completion of the catalytic cycle.
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Determining the dimensions of the Drug-Binding domain of human P-glycoprotein using thiol cross-linking compounds as molecular rulers.
Journal of Biological Chemistry, 2001Co-Authors: David M ClarkeAbstract:Abstract The human multiDrug resistance P-glycoprotein (P-gp) interacts with a broad range of compounds with diverse structures and sizes. There is considerable evidence indicating that residues in transmembrane segments 4–6 and 10–12 form the Drug-Binding Site. We attempted to measure the size of the Drug-Binding Site by using thiol-specific methanethiosulfonate (MTS) cross-linkers containing spacer arms of 2 to 17 atoms. The majority of these cross-linkers were also substrates of P-gp, because they stimulated ATPase activity (2.5- to 10.1-fold). 36 P-gp mutants with pairs of cysteine residues introduced into transmembrane segments 4–6 and 10–12 were analyzed after reaction with 0.2 mm MTS cross-linker at 4 °C. The cross-linked product migrated with lower mobility than native P-gp in SDS gels. 13 P-gp mutants were cross-linked by MTS cross-linkers with spacer arms of 9–25 A. Vinblastine and cyclosporin A inhibited cross-linking. The emerging picture from these results and other studies is that the Drug-Binding domain is large enough to accommodate compounds of different sizes and that the Drug-Binding domain is “funnel” shaped, narrow at the cytoplasmic side, at least 9–25 A in the middle, and wider still at the extracellular surface.
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defining the Drug Binding Site in the human multiDrug resistance p glycoprotein using a methanethiosulfonate analog of verapamil mts verapamil
Journal of Biological Chemistry, 2001Co-Authors: David M ClarkeAbstract:Abstract Defining the residues involved in the Binding of a substrate provides insight into how the human multiDrug resistance P-glycoprotein (P-gp) can transport a wide range of structurally diverse compounds out of the cell. Because verapamil is the most potent stimulator of P-gp ATPase activity, we synthesized a thiol-reactive analog of verapamil (MTS-verapamil) and used it with cysteine-scanning mutagenesis to identify the reactive residues within the Drug-Binding domain of P-gp. MTS-verapamil stimulated the ATPase activity of Cys-less P-gp and had a K m value (25 μm) that was similar to that of verapamil. 252 P-gp mutants containing a single cysteine within the predicted transmembrane (TM) segments were expressed in HEK 293 cells and purified by nickel-chelate chromatography and assayed for inhibition by MTS-verapamil. The activities of 15 mutants, Y118C (TM2), V125C (TM2), S222C (TM4), L339C (TM6), A342C (TM6), A729C (TM7), A841C (TM9), N842C (TM9), I868C (TM10), A871C (TM10), F942C (TM11), T945C (TM11), V982C (TM12), G984C (TM12), and A985C (TM12), were inhibited by MTS-verapamil. Four mutants, S222C (TM4), L339C (TM6), A342C (TM6), and G984C (TM12), were significantly protected from inhibition by MTS-verapamil by pretreatment with verapamil. Less protection was observed in mutants I868C (TM10), F942C (TM11) and T945C (TM11). These results indicate that residues in TMs 4, 6, 10, 11, and 12 must contribute to the Binding of verapamil.
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identification of residues in the Drug Binding Site of human p glycoprotein using a thiol reactive substrate
Journal of Biological Chemistry, 1997Co-Authors: David M ClarkeAbstract:Abstract We identified a thiol-reactive compound, dibromobimane (dBBn), that was a potent stimulator (8.2-fold) of the ATPase activity of Cys-less P-glycoprotein. We then used this compound together with cysteine-scanning mutagenesis to identify residues in transmembrane segment (TM) 6 and TM12 that are important for function. TM6 and TM12 lie close to each other in the tertiary structure and are postulated to be important for Drug-protein interactions. The majority of P-glycoprotein mutants containing a single cysteine residue retained substantial amounts of Drug-stimulated ATPase activity and were not inhibited by dBBn. The ATPase activities of mutants L339C, A342C, L975C, V982C, and A985C, however, were markedly inhibited (>60%) by dBBn. The Drug substrates verapamil, vinblastine, and colchicine protected these mutants against inhibition by dBBn, suggesting that these residues are important for interaction of substrates with P-glycoprotein. We previously showed that residues Leu339, Ala342, Leu975, Val982, and Ala985 lie along the point of contact between helices TM6 and TM12, when both are aligned in a left-handed coiled coil (Loo, T. W., and Clarke, D. M. (1997)J. Biol. Chem. 272, 20986–20989). Taken together, these results suggest that the interface between TM6 and TM12 likely forms part of the potential Drug-Binding pocket in P-glycoprotein.
Peter Lukacs - One of the best experts on this subject based on the ideXlab platform.
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the enigmatic Drug Binding Site for sodium channel inhibitors
Current Molecular Pharmacology, 2010Co-Authors: Arpad Mike, Peter LukacsAbstract:: Local anesthetics have been in clinical use since 1884, and different aspects of the local anesthetic Binding Site have been studied in enormous detail. In spite of all these efforts, some of the most fundamental questions--such as which exact residues constitute the Binding Site, how many Binding Sites exist, do local anesthetics share their Binding Site(s) with other sodium channel inhibitors, and what are the mechanisms of inhibition--are still largely unanswered. We review accumulated data on the "local anesthetic receptor"and discuss controversial points, such as possible mechanisms of inhibition, the possibility of additional Binding Sites, the orientation of S6 helices, and the internal vs. external position of the anticonvulsant Binding Site. We describe the four following specific groups of functionally important residues: i) conserved asparagines six residues below the hinge residues; we propose that they are oriented toward the external surface of S6 helices, and have a critical role in the coupling of voltage sensors to gating, ii) residues lining the inner vestibule and constructing the "orthodox" Binding Site, iii) residues around the outer vestibule, which have been proposed to constitute an alternative external Binding Site, and iv) residues determining external access for quaternary amine inhibitors, such as QX314. We conclude that sodium channel inhibitors must be heterogenous in terms of Binding Sites and inhibition mechanisms, and propose that this heterogeneity should be taken into consideration during Drug development.
Frances J Sharom - One of the best experts on this subject based on the ideXlab platform.
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interaction of lds 751 with the Drug Binding Site of p glycoprotein a trp fluorescence steady state and lifetime study
Archives of Biochemistry and Biophysics, 2009Co-Authors: Miguel R Lugo, Frances J SharomAbstract:Abstract P-glycoprotein (ABCB1) is an ATP-driven efflux pump which binds Drugs within a large flexible Binding pocket. Intrinsic Trp fluorescence was used to probe the interactions of LDS-751 (2-[4-(4-[dimethylamino]phenyl)-1,3-butadienyl]-3-ethylbenzo-thiazolium perchlorate) with purified P-glycoprotein, using steady-state/lifetime measurements and collisional quenching. The fast decay component of P-glycoprotein intrinsic fluorescence (τ1 = 0.97 ns) was unaffected by LDS-751 Binding, while the slow decay component (τ2 = 4.02 ns) was quenched by dynamic and static mechanisms. Both the wavelength-dependence of the decay kinetics, and the time-resolved emission spectra, suggested the existence of excited-state relaxation processes within the protein matrix on the nanosecond time-scale, which were altered by LDS-751 Binding. The fast decay component, which is more solvent-exposed, can be attributed to cytosolic/extracellular Trp residues, while the slow decay component likely arises from more buried transmembrane Trp residues. Interaction of a Drug with the Binding pocket of P-glycoprotein thus affects its molecular structure and fast dynamics.
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interaction of lds 751 with p glycoprotein and mapping of the location of the r Drug Binding Site
Biochemistry, 2005Co-Authors: Miguel R Lugo, Frances J SharomAbstract:One cause of multiDrug resistance is the overexpression of P-glycoprotein, a 170 kDa plasma membrane ABC transporter, which functions as an ATP-driven efflux pump with broad specificity for hydrophobic Drugs, peptides, and natural products. The protein appears to interact with its substrates within the membrane environment. Previous reports suggested the existence of at least two Binding Sites, possibly overlapping and displaying positively cooperative interactions, termed the H and R Sites for their preference for Hoechst 33342 and rhodamine 123, respectively. In this work, we have used several fluorescence approaches to characterize the molecular interaction of purified P-glycoprotein (Pgp) with the dye LDS-751, which is proposed to bind to the R Site. A 50-fold enhancement of LDS-751 fluorescence indicated that the protein Binding Site was located in a hydrophobic environment, with a polarity lower than that of chloroform. LDS-751 bound with sub-micromolar affinity (Kd ) 0.75 IM) and quenched P-glycoprotein intrinsic Trp fluorescence by 40%, suggesting that Trp emitters are probably located close to the drub-Binding regions of the transporter and may interact directly with the dye. Using a FRET approach, we mapped the possible locations of the LDS-751 Binding Site relative to the NB domain active Sites. The R Site appeared to be positioned close to the membrane boundary of the cytoplasmic leaflet. The location of both H and R Drug Binding Sites is in agreement with the idea that Pgp may operate as a Drug flippase, moving substrates from the inner leaflet to the outer leaflet of the plasma membrane. 1 Abbreviations: ABC, ATP-Binding cassette; AMP-PNP, 5'-ade- nylylimidodiphosphate; CHAPS, 3-((3-cholamidopropyl)dimethylam- monio)-1-propanesulfonate; DMSO, dimethyl sulfoxide; H33342, Ho- echst 33342; FRET, fluorescence resonance energy transfer; GuHCl, guanidine hydrochloride; LDS-751, 2-{4-(4-(dimethylamino)phenyl)- 1,3-butadienyl}-3-ethylbenzothiazolium perchlorate; MDR, multiDrug resistant or resistance; MIANS, 2-(4-maleimidoanilino)naphthalene-6- sulfonic acid; NATA, N-acetyltryptophanamide; NB, nucleotide bind- ing; NBD-Cl, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole; Pgp, P-glyco- protein; TM, transmembrane; TNP-ATP, 2'(3')-O-(2,4,6-triphenyl)- adenosine 5'-triphosphate. 643
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proximity of bound hoechst 33342 to the atpase catalytic Sites places the Drug Binding Site of p glycoprotein within the cytoplasmic membrane leaflet
Biochemistry, 2002Co-Authors: Qin Qu, Frances J SharomAbstract:: The P-glycoprotein multiDrug transporter carries out ATP-driven cellular efflux of a wide variety of hydrophobic Drugs, natural products, and peptides. Multiple Binding Sites for substrates appear to exist, most likely within the hydrophobic membrane spanning regions of the protein. Since ATP hydrolysis is coupled to Drug transport, the spatial relationship of the Drug Binding Sites relative to the ATPase catalytic Sites is of considerable interest. We have used a fluorescence resonance energy transfer (FRET) approach to estimate the distance between a bound substrate and the catalytic Sites in purified P-glycoprotein. The fluorescent dye Hoechst 33342 (H33342), a high-affinity P-glycoprotein substrate, bound to the transporter and acted as a FRET donor. H33342 showed greatly enhanced fluorescence emission when bound to P-glycoprotein, together with a substantial blue shift, indicating that the Drug Binding Site is located in a nonpolar environment. Cys428 and Cys1071 within the catalytic Sites of P-glycoprotein were covalently labeled with the acceptor fluorophore NBD-Cl (7-chloro-4-nitrobenz-2-oxa-1,3-diazole). H33342 fluorescence was highly quenched when bound to NBD-labeled P-glycoprotein relative to unlabeled protein, indicating that FRET takes place from the bound dye to NBD. The distance separating the bound dye from the NBD acceptor was estimated to be approximately 38 A. Transition-state P-glycoprotein with the complex ADP*orthovanadate*Co2+ stably trapped at one catalytic Site bound H33342 with similar affinity, and FRET measurements led to a similar separation distance estimate of 34 A. Since previous FRET studies indicated that a fluorophore bound within the catalytic Site was positioned 31-35 A from the interfacial region of the bilayer, the H33342 Binding Site is likely located 10-14 A below the membrane surface, within the cytoplasmic leaflet of the membrane, in both resting-state and transition-state P-glycoprotein.
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the p glycoprotein efflux pump how does it transport Drugs
The Journal of Membrane Biology, 1997Co-Authors: Frances J SharomAbstract:: Pgp is an atypical translocating ATPase, with low affinity for ATP and high constitutive ATPase activity. Pgp also has an unusually broad specificity for hydrophobic substrates, including many chemotherapeutic Drugs. Transport studies in reconstituted systems indicate that Drug transport requires ATP hydrolysis and is active, generating a Drug concentration gradient. Binding of Drugs and ATP to Pgp induces conformational changes in the protein, and the Drug Binding Site is conformationally coupled to the NBDs. Evidence accumulated to date suggests that the transporter interacts directly with nonpolar substrates within the membrane environment, and may act as a Drug flippase, moving Drugs from the inner to the outer leaflet of the bilayer. Chemosensitizers that block the action of Pgp are proposed to act as alternative substrates, but their high rate of spontaneous flip-flop across the membrane results in futile cycling of the transporter.
