Identification of the Distance between the Homologous Halves of P-glycoprotein That Triggers the High/Low ATPase Activity Switch

Loo, Tip W.; Clarke, David M.

doi:10.1074/jbc.m114.552075

Cited by 19 publications

(15 citation statements)

References 69 publications

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“…Consistently, our ⌬lnk mP-gp mutant has an elevated basal ATPase activity (Fig. 1A), and cross-linking between ICDs also led to higher ATPase activity (51).…”

Section: Coupling Of Open-and-close Motion Of P-gp Two Halves To Movesupporting

confidence: 61%

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

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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“…Consistently, our ⌬lnk mP-gp mutant has an elevated basal ATPase activity (Fig. 1A), and cross-linking between ICDs also led to higher ATPase activity (51).…”

Section: Coupling Of Open-and-close Motion Of P-gp Two Halves To Movesupporting

confidence: 61%

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

165

161

View full text Add to dashboard Cite

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“…By contrast, cross-linking of the intracellular cysteines resulted in stimulation of ATPase activity. Direct cross-linking of the cysteines L175C and N820C (23) or cross-linking of mutant P571C/I1050C with the short 1,4-butanediyl bismethanethiosulfonate cross-linker (7.8 Å) (24) highly activated P-gp ATPase activity over 10-fold in the absence of drug substrates. Cysteines L175C/N820C are located in the intracellular loops (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The cells were then harvested and washed with PBS (pH 7.4) containing 5 mM DTT to reduce the spontaneously formed disulfide bond. Membranes were prepared as described previously (23) and suspended in TBS (pH 7.4) with or without 5 mM ATP plus 10 mM MgCl 2 in an ice bath (0°C). The samples were then treated for 5 min at 0°C with or without 1 mM copper phenanthroline.…”

Section: Construction and Expression Of Mutants-mentioning

confidence: 99%

“…We showed previously that alanine replacements of the seven endogenous cysteines yielded an active Cys-less P-gp (36). Cross-linking can be readily detected because disulfide bonds between the halves will cause the protein to migrate more slowly on SDS-PAGE gels (23).…”

Section: Cysteines Introduced Into Ecl 1 (A80c) and Ecl 4 (R741c) Ofmentioning

confidence: 99%

“…1B). We showed previously that disulfide cross-linking could trap P-gp in a closed conformation to activate its ATPase activity (23)(24)(25).…”

mentioning

confidence: 99%

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Cysteines Introduced into Extracellular Loops 1 and 4 of Human P-Glycoprotein That Are Close Only in the Open Conformation Spontaneously Form a Disulfide Bond That Inhibits Drug Efflux and ATPase Activity

Loo

Clarke

2014

Journal of Biological Chemistry

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Background: P-glycoprotein (P-gp) may alternate between the inward-facing (open) or outward-facing (closed) conformations during the reaction cycle. Results: Cysteines introduced into extracellular loops 1 and 4 cross-linked spontaneously and inhibited drug efflux and ATPase activity. Conclusion: P-gp activity was inhibited when P-gp was trapped in an open conformation. Significance: P-gp can be blocked by multiple mechanisms involving extracellular loops.

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Structures and Transport Mechanisms of the ABC Efflux Pumps

Orelle

Jault

2016

Efflux-Mediated Antimicrobial Resistance in Bacteria

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Identification of the Distance between the Homologous Halves of P-glycoprotein That Triggers the High/Low ATPase Activity Switch

Cited by 19 publications

References 69 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

Cysteines Introduced into Extracellular Loops 1 and 4 of Human P-Glycoprotein That Are Close Only in the Open Conformation Spontaneously Form a Disulfide Bond That Inhibits Drug Efflux and ATPase Activity

Structures and Transport Mechanisms of the ABC Efflux Pumps

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