Structure of ABCB1/P-Glycoprotein in the Presence of the CFTR Potentiator Ivacaftor

Barbieri, Alessandro; Thonghin, Nopnithi; Shafi, Talha; Prince, Stephen M.; Collins, Richard F.; Ford, Robert C.

doi:10.3390/membranes11120923

Cited by 18 publications

(17 citation statements)

References 60 publications

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“…The primary differences between the two structures of inward-facing conformations of At Atm3 are in the relative positioning of the NBDs which are more widely separated in the apo structure relative to the GSSG-bound structure. Similar substrate-induced NBD movements have been previously observed in MRP1 ( Johnson and Chen, 2017 ) and ABCB1 ( Barbieri et al, 2021 ).…”

Section: Discussionsupporting

confidence: 84%

Glutathione binding to the plant AtAtm3 transporter and implications for the conformational coupling of ABC transporters

Fan

Rees

2022

eLife

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The ATP Binding Cassette (ABC) transporter of mitochondria (Atm) from Arabidopsis thaliana (AtAtm3) has been implicated in the maturation of cytosolic iron-sulfur proteins and heavy metal detoxification, plausibly by exporting glutathione derivatives. Using single-particle cryo-electron microscopy, we have determined four structures of AtAtm3 in three different conformational states: two inward-facing conformations (with and without bound oxidized glutathione (GSSG)), together with closed and outward-facing states stabilized by MgADP-VO4. These structures not only provide a structural framework for defining the alternating access transport cycle, but also reveal the paucity of cysteine residues in the glutathione binding site that could potentially form inhibitory mixed disulfides with GSSG. Despite extensive efforts, we were unable to prepare the ternary complex of AtAtm3 containing both GSSG and MgATP. A survey of structurally characterized type IV ABC transporters that includes AtAtm3 establishes that while nucleotides are found associated with all conformational states, they are effectively required to stabilize occluded, closed, and outward-facing conformations. In contrast, transport substrates have only been observed associated with inward-facing conformations. The absence of structures with dimerized nucleotide binding domains containing both nucleotide and transport substrate suggests that this form of the ternary complex exists only transiently during the transport cycle.

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Section: Discussionsupporting

confidence: 84%

Glutathione binding to the plant AtAtm3 transporter and implications for the conformational coupling of ABC transporters

Fan

Rees

2022

eLife

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“…20 To verify that multiplexed copolymers of QN produced using free radical copolymerization retain the ability of QN to bind P-gp, an in vitro growth study was performed. Because P-gp is a membrane-bound protein, indirect techniques, such as in vitro growth assays, are often used to characterize binding interactions. , Typically, cells expressing P-gp are maintained in culture and treated with a known P-gp substrate, and changes in substrate efflux are used as a measure of P-gp binding to a test material …”

Section: Resultsmentioning

confidence: 99%

Selective Accumulation of Near Infrared-Labeled Multivalent Quinidine Copolymers in Tumors Overexpressing P-Glycoprotein: Potential for Noninvasive Diagnostic Imaging

Robertus

Snyder

Curley

et al. 2023

ACS Appl. Bio Mater.

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P-glycoprotein (P-gp) is a promiscuous small molecule transporter whose overexpression in cancer is associated with multidrug resistance (MDR). In these instances, anticancer drugs can select for P-gp-overexpressing cells, leading to cancer recurrence with an MDR phenotype. To avoid selection for MDR cancers and inform individual patient treatment plans, it is critical to noninvasively identify P-gp-overexpressing tumors prior to administration of chemotherapy. We report the facile free radical copolymerization of quinidine, a competitive inhibitor of P-gp, and acrylic acid to generate multiplexed polymeric P-gp-targeted imaging agents with tunable quinidine content. Copolymer targeting was demonstrated in a nude mouse xenograft model. In xenografts overexpressing P-gp, copolymer distribution was enhanced over two-fold compared to the negative control of poly(acrylic acid) regardless of quinidine content. In contrast, accumulation of the copolymers in xenografts lacking P-gp was equivalent to poly(acrylic acid). This work forms the foundation for a unique approach toward the phenotype-specific noninvasive imaging of MDR tumors and is the first in vivo demonstration of copolymer accumulation through the active targeting of P-gp.

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“…AFM simulations were performed on Pgp structures with the nucleotide-binding domains (NBDs) separated in the IF conformation and in the OF conformation. The apo state of Pgp has a wide dynamic range, but evidence suggests that the majority of apo Pgp resides in the IF conformation, in which the NBDs are separated 32,39 . The apo conformation is simulated as before, and the OF conformation is simulated using one of the few structures of this state (PDB: 6C0V 40 Figure 2A shows that C-side heights range from 5.7 to 8.7 nm, consistent with Pgp shifting between the IF and OF conformations 32,39 .…”

Section: Resultsmentioning

confidence: 99%

“…The apo state of Pgp has a wide dynamic range, but evidence suggests that the majority of apo Pgp resides in the IF conformation, in which the NBDs are separated 32,39 . The apo conformation is simulated as before, and the OF conformation is simulated using one of the few structures of this state (PDB: 6C0V 40 Figure 2A shows that C-side heights range from 5.7 to 8.7 nm, consistent with Pgp shifting between the IF and OF conformations 32,39 . Of the individual protrusions we imaged, the largest percentage (~ 41%) exhibited a height of 6.9 ± 0.5nm (Fig.…”

Section: Resultsmentioning

confidence: 99%

AFM kymographs can provide robust conformational transition detection for pharmaceutically relevant membrane proteins in lipid bilayers

Schaefer

Roberts²,

King

2023

Preprint

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Membrane proteins play critical roles in disease and in the disposition of many pharmaceuticals. A prime example is P-glycoprotein (Pgp) which moves a diverse range of drugs across membranes and out of the cell before a therapeutic payload can be delivered. Conventional structural biology methods have provided a valuable framework for comprehending the complex conformational changes underlying Pgp function, which also includes ATPase activity, but the lack of real-time information hinders understanding. Atomic force microscopy (AFM) is a single-molecule technique that is well-suited for studying active membrane proteins in bilayers and is poised to advance the field beyond static snapshots. After verifying Pgp activity in surface-support bilayers, we used kymograph analysis in conjunction with AFM imaging and simulations to study structural transitions at the 100 ms timescale. Though kymographs are frequently employed to boost temporal resolution, the limitations of the method have not been well characterized, especially for sparse non-crystalline distributions of pharmaceutically relevant membrane proteins like Pgp. Common experimental challenges are analyzed, including protein orientation, instrument noise, and drift. Surprisingly, a lateral drift of 75% of the protein dimension leads to only a 12% probability of erroneous state transition detection; average dwell time error achieves a maximum value of 6%. Rotational drift of proteins like Pgp, with azimuthally-dependent maximum heights, can lead to artifactual transitions. This pitfall may be alleviated by torsional constraints. Confidence in detected transitions may be increased by using conformation-altering ligands such as non-hydrolysable analogs. Overall, the data indicate that AFM kymographs are a viable method to access conformational dynamics for Pgp, but generalizations of the method should be made with caution.

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Structure of ABCB1/P-Glycoprotein in the Presence of the CFTR Potentiator Ivacaftor

Cited by 18 publications

References 60 publications

Glutathione binding to the plant AtAtm3 transporter and implications for the conformational coupling of ABC transporters

Glutathione binding to the plant AtAtm3 transporter and implications for the conformational coupling of ABC transporters

Selective Accumulation of Near Infrared-Labeled Multivalent Quinidine Copolymers in Tumors Overexpressing P-Glycoprotein: Potential for Noninvasive Diagnostic Imaging

AFM kymographs can provide robust conformational transition detection for pharmaceutically relevant membrane proteins in lipid bilayers

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