A single active catalytic site is sufficient to promote transport in P-glycoprotein

Bársony, Orsolya; Szalóki, Gábor; Türk, Dóra; Tarapcsák, Szabolcs; Gutay-Tóth, Zsuzsanna; Bacsó, Zsolt; Holb, I. J.; Székvölgyi, Lóránt; Szabó, Gábor; Csanády, László; Szakács, Gergely; Goda, Katalin

doi:10.1038/srep24810

Cited by 42 publications

(46 citation statements)

References 71 publications

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“…We next wanted to explore inter-domain communication between the TMD and the NBD of the DrrAB complex. The first evidence of such communication was obtained through dual titration experiments, where prior binding of ATP resulted in reduced affinity for Dox or vinblastine, as also observed previously with nucleotide-bound Pgp [58–61] and other ABC transporters [36, 62–64]. This observation not only suggests cross-talk between DrrA and DrrB, but it also indicates that ATP binding may act as a power stroke for switching the conformation of DrrB from inward-facing to outward-facing resulting in release of the drug to the outside.…”

Section: Discussionmentioning

confidence: 62%

Conformational changes in a multidrug resistance ABC transporter DrrAB: Fluorescence-based approaches to study substrate binding

Rahman

Kaur

2018

Archives of Biochemistry and Biophysics

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Bacterial multidrug transporter DrrAB exhibits overlapping substrate specificity with mammalian Pglycoprotein. DrrA hydrolyzes ATP, and the energy is transduced to carrier DrrB resulting in export of drugs. Previous studies suggested that DrrB contains a large and flexible drug-binding pocket made of aromatic residues contributed by several transmembrane helices with different drugs binding to both specific and shared residues in this pocket. However, direct binding of drugs to DrrAB or the mechanism of substrate-induced conformational changes between DrrA and DrrB has so far not been investigated. We used two fluorescence-based approaches to determine substrate binding to purified DrrAB. Our analysis shows that DrrB binds drugs with variable affinities and contains multiple drug binding sites. This work also provides evidence for two asymmetric nucleotide binding sites in DrrA with strikingly different binding affinities. Using targeted fluorescence labeling, we provide clear evidence of long-range conformational changes occurring between DrrA and DrrB. It is proposed that the transduction pathway from the nucleotide-binding DrrA subunit to the substrate binding DrrB subunit includes Q-loop and CREEM motifs in DrrA and EAA-like motif in DrrB. This study lays a solid groundwork for examining roles of various conserved regions of DrrA and DrrB in transduction of conformational changes.

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Section: Discussionmentioning

confidence: 62%

Conformational changes in a multidrug resistance ABC transporter DrrAB: Fluorescence-based approaches to study substrate binding

Rahman

Kaur

2018

Archives of Biochemistry and Biophysics

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“…The results for BD-verapamil and BD-vinblastine support the well-known behavior of P-gp, wherein the equilibrium affinity of drugs may be decreased in the presence of nucleotides, but the magnitude of the decrease in affinity can be drug-dependent and nucleotide-dependent. Addition of AMP-PNP to the complex of BD-vinblastine with human P-gp is sufficient to “release” drug, 39 but here the nucleotide causes an increase in K D for BD-vinblastine of only 2-fold.…”

Section: Discussionmentioning

confidence: 97%

Differential Coupling of Binding, ATP Hydrolysis, and Transport of Fluorescent Probes with P-Glycoprotein in Lipid Nanodiscs

Nath

Atkins

2017

Biochemistry

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The ATP binding cassette transporter P-glycoprotein (ABCB1 or P-gp) plays a major role in cellular resistance to drugs and drug interactions. Experimental studies support a mechanism with nucleotide-dependent fluctuation between inward-facing and outward-facing conformations, which are coupled to nucleotide hydrolysis. However, detailed insight into drug-dependent modulation of these conformational ensembles is lacking. Different drugs likely occupy partially overlapping but distinct sites and are therefore variably coupled to nucleotide binding and hydrolysis. Many fluorescent drug analogues are used in cell-based transport models; however, their specific interactions with P-gp have not been studied, and this limits interpretation of transport assays in terms of molecular models. Here we monitor binding of the fluorescent probe substrates BODIPY-verapamil, BODIPY-vinblastine, and Flutax-2 at low occupancy to murine P-gp in lipid nanodiscs via fluorescence correlation spectroscopy, in variable nucleotide-bound states. Changes in affinity for the different nucleotide-dependent conformations are probe-dependent. For BODIPY-verapamil and BODIPY-vinblastine, there are 2–10-fold increases in KD in the nucleotide-bound or vanadate-trapped state, compared to that in the nucleotide-free state. In contrast, the affinity of Flutax-2 is unaffected by nucleotide or vanadate trapping. In further contrast to BODIPY-verapamil and BODIPY-vinblastine, Flutax-2 does not cause stimulation of ATP hydrolysis despite the fact that it is transported in vesicle-based transport assays. Whereas the established substrates verapamil, paclitaxel, and vinblastine displace BODIPY-verapamil or BODIPY-vinblastine from their high-affinity sites, the transport substrate Flutax-2 is not displaced by any of these substrates. The results demonstrate a unique binding site for Flutax-2 that allows for transport without stimulation of ATP hydrolysis.

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“…Accordingly, we mutated tyrosine residues Y310 and Y953 to alanine and determined rh123 steady‐state accumulation and zero‐trans efflux rates in these two single mutants. The E556Q mutant (harboring a mutated catalytic glutamate in the Walker B motif of NBS1), mock transfected cells, and the Walker A double mutant K433M/K1076M served as negative controls. In these three negative controls, comparable steady‐state accumulation levels of approximately 1000 MFU/cell were found.…”

Section: Resultsmentioning

confidence: 99%

Active transport of rhodamine 123 by the human multidrug transporter P‐glycoprotein involves two independent outer gates

Nasim

Schmid

Sohail

et al. 2020

Pharmacology Res & Perspec

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Human P-glycoprotein (P-gp) is a multispecific drug-efflux transporter, which plays an important role in drug resistance and drug disposition. Recent cryo-electron microscopy structures confirmed its rotationally symmetric architecture, which allows dual interaction with ATP and substrates. We here report the existence of two distinct, symmetry-related outer gates. Experiments were aided by availability of the X-ray structure of a homodimeric eukaryotic homolog of P-gp from red alga (CmABCB1), which defined the role of an apical tyrosine residue (Y358) in outer gate formation.We mutated analogous tyrosine residues in each half of the human full-length transporter (Y310, Y953) to alanine. These mutants were introduced in engineered transporters which bind rhodamine 123 in one of two symmetry-related binding modes only. Outer gate dysfunction was detected by a loss of active transport characteristics, while these mutants retained the ability for outward downhill transport. Our data demonstrate that symmetric tyrosine residues Y310 and Y953 are involved in formation of two distinct symmetry-related outer gates, which operate contingent on the rhodamine 123 binding mode. Hence, the rotationally symmetric architecture of P-gp, which determines duality in ATP binding and rhodamine 123 interaction, also forms the basis for the existence of two independently operating outer gates. K E Y W O R D S ABC transporter, ABCB1, functional biology, multidrug transporter, outer gates, P-glycoprotein S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Nasim F, Schmid D, Szakács G, et al. Active transport of rhodamine 123 by the human multidrug transporter P-glycoprotein involves two independent outer gates. Pharmacol Res Perspect. 2020;00:e00572. https://doi.

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A single active catalytic site is sufficient to promote transport in P-glycoprotein

Cited by 42 publications

References 71 publications

Conformational changes in a multidrug resistance ABC transporter DrrAB: Fluorescence-based approaches to study substrate binding

Conformational changes in a multidrug resistance ABC transporter DrrAB: Fluorescence-based approaches to study substrate binding

Differential Coupling of Binding, ATP Hydrolysis, and Transport of Fluorescent Probes with P-Glycoprotein in Lipid Nanodiscs

Active transport of rhodamine 123 by the human multidrug transporter P‐glycoprotein involves two independent outer gates

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