Mutations in Intracellular Loops 1 and 3 Lead to Misfolding of Human P-glycoprotein (ABCB1) That Can Be Rescued by Cyclosporine A, Which Reduces Its Association with Chaperone Hsp70

Kapoor, Khyati; Bhatnagar, Jaya; Chufán, Eduardo E.; Ambudkar, Suresh V.

doi:10.1074/jbc.m113.498980

Cited by 22 publications

(22 citation statements)

References 44 publications

(59 reference statements)

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“…3, A and B). While in contact with the amine group of the adenine ring, these two residues do not appear to have strong interactions with the bound ATP, which is consistent with the phenotype displayed by D164A or D164C mutations in human P-gp (33). Instead, they form a hydrogen bonding network with Tyr-440 of NBD1.…”

Section: Restricting Conformational Flexibility Leads To Dysfunction supporting

confidence: 54%

Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity

Esser

Zhou

Pluchino

et al. 2017

Journal of Biological Chemistry

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165

161

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Edited by Norma AllewellP-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing motion of the two halves of P-gp. The open-andclose motion alters the surface topology of P-gp within the drugbinding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions.The association of multidrug resistance (MDR) 3 in cancer treatment with expression of human ABC transporters on the cell surface has raised the possibility that overcoming MDR might be achieved by inhibiting these transporters (1). One transporter in particular, the human P-glycoprotein (P-gp), has been well characterized; it is able to confer MDR by transporting numerous structurally unrelated drugs at the expense of hydrolyzing ATP (2, 3). In addition, P-gp plays important roles in drug distribution in normal physiology and is an essential component of many physiological barriers (4, 5).Long-standing efforts have been devoted to understanding the mechanism of P-gp function by various experimental approaches. Among them, structural studies of P-gp from various organisms have been reported (6 -8). In particular, a number of structures for the mouse P-gp (mP-gp) were obtained (8 -10). However, the diffraction limits of "near-native," inhibitor-bound, and nanobody-associated mP-gp crystals were relatively low, mostly near 4 Å resolution. The methylated protein gave higher resolution structures (11), but the impact of reductive methylation on the structure and function of P-gp was not addressed. The low resolution diffraction of P-gp crystals has been attributed in part to the intrinsic flexibility of the molecule (12-14).Mouse P-gp is a 1276-residue polypeptide and bears 87% sequence identity to human P-gp; it consists of two homologous halves connected by a flexible linker of ϳ75 residues. Each half is made of a transmembrane domain (TMD) implicated in drug recognition and transport and a nucleotide-binding domain (NBD). Each NBD is able to bind and hydrolyze ATP (15, 16), which is, however, dependent on the other NBD being functional (17,18). The two NBDs of P-gp are highly homologous, each featuring a consensus nucleotide-binding site with full-fledged Walker...

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Section: Restricting Conformational Flexibility Leads To Dysfunction supporting

confidence: 54%

Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity

Esser

Zhou

Pluchino

et al. 2017

Journal of Biological Chemistry

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165

161

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“…The molecular simulation study (34) sug- . In support of this prediction, it has been reported recently that a P-gp mutant containing the D805C mutation reduced verapamil-stimulated ATPase activity by about 70% but had no effect on the K m for ATP (49).…”

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confidence: 58%

Locking Intracellular Helices 2 and 3 Together Inactivates Human P-glycoprotein

Loo

Clarke

2014

Journal of Biological Chemistry

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Background: P-glycoprotein is an ATP-dependent drug pump. Results: Mutations or cross-linking of intracellular loops (ICL) 2 and 3 inhibit folding and/or activity. Conclusion: Hydrophobic residues in ICL2 and ICL3 and the second nucleotide-binding domain form a hydrophobic transmission network for folding and activity. Significance: We identify a dual-purpose transmission interface required for folding and activity.

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“…Note glycosylation in wild-type, chimeric, and codon-optimized P-gp are not observed, as insect cells do not glycosylate human or mouse P-gp. 37 …”

Section: Resultsmentioning

confidence: 99%

Human–Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P-Glycoprotein (ABCB1)

et al. 2016

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The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter's broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human-mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-crosslinking with [125I]-Iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug-binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes.

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Mutations in Intracellular Loops 1 and 3 Lead to Misfolding of Human P-glycoprotein (ABCB1) That Can Be Rescued by Cyclosporine A, Which Reduces Its Association with Chaperone Hsp70

Cited by 22 publications

References 44 publications

Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity

Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity

Locking Intracellular Helices 2 and 3 Together Inactivates Human P-glycoprotein

Human–Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P-Glycoprotein (ABCB1)

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