Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates

Ghosh, Pratiti; Moitra, Karobi; Maki, Nazli; Dey, Saikat

doi:10.1016/j.abb.2006.02.025

Cited by 11 publications

(8 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We investigated the role of the linker region in human P‐gp that spans from approximately Glu633 to Tyr709. As previously reported [21–24], the linker region appears to be the most flexible part of the P‐gp structure (Fig. 1).…”

Section: Discussionsupporting

confidence: 81%

“…To investigate the role of the linker region of P-gp, a linker-cleaved P-gp was generated by protease cleavage because the linker region is highly susceptible to protease cleavage [21][22][23][24]. Highly purified P-gp in detergent micelles was incubated with trypsin, chymotrypsin, V8 protease, or subtilisin, as indicated in the Materials and methods.…”

Section: Protease Treatment Of Purified P-gp In Detergent Micellesmentioning

confidence: 99%

“…Recently, limited proteolysis of the linker region has shed light on its involvement in the ATPase activity of P‐gp. The linker region has been shown to be highly susceptible to different proteases [21–24], and the trypsin and chymotrypsin cleavage sites were identified as Arg680 and Leu682, respectively [22]. Trypsin cleavage increased the basal, verapamil‐stimulated and vinblastine‐stimulated ATPase activities, suggesting that cleavage of the linker activates P‐gp [21].…”

mentioning

confidence: 99%

See 2 more Smart Citations

Functional role of the linker region in purified human P‐glycoprotein

et al. 2009

View full text Add to dashboard Cite

Human P-glycoprotein (P-gp, ABCB1), which conveys multidrug resistance, is a drug efflux pump that transports a wide variety of structurally unrelated compounds out of cells [1][2][3][4]. The transport by P-gp is driven by energy from ATP hydrolysis, and P-gp is classified as a member of the ATP-binding cassette (ABC) transporter family [5,6].The transport of substrate by P-gp is thought to be coupled with ATP hydrolysis [7]. Without a transport substrate, P-gp has low basal ATP hydrolase (ATPase) activity, whereas with substrates P-gp exhibits high ATPase activity, which is known as substrate-stimulated ATPase activity [8][9][10][11]. Thus, the substrate-stimulated ATPase activity can be a measure of the recognition of substrate by P-gp. When titrating P-gp substrates, the activity increases up to a maximum but then decreases at high substrate concentrations. This characteristic bell-shaped activity profile has been Human P-glycoprotein (P-gp), which conveys multidrug resistance, is an ATP-dependent drug efflux pump that transports a wide variety of structurally unrelated compounds out of cells. P-gp possesses a 'linker region' of $ 75 amino acids that connects two homologous halves, each of which contain a transmembrane domain followed by a nucleotide-binding domain. To investigate the role of the linker region, purified human P-gp was cleaved by proteases at the linker region and then compared with native P-gp. Based on a verapamil-stimulated ATP hydrolase assay, sizeexclusion chromatography analysis and a thermo-stability assay, cleavage of the P-gp linker did not directly affect the preservation of the overall structure or the catalytic process in ATP hydrolysis. However, linker cleavage increased the k cat values both with substrate (k sub ) and without substrate (k basal ), but decreased the k sub ⁄ k basal values of all 10 tested substrates. The former result indicates that cleaving the linker activates P-gp, while the latter result suggests that the linker region maintains the tightness of coupling between the ATP hydrolase reaction and substrate recognition. Inspection of structures of the P-gp homolog, MsbA, suggests that linker-cleaved P-gp has increased ATP hydrolase activity because the linker interferes with a conformational change that accompanies the ATP hydrolase reaction. Moreover, linker cleavage affected the specificity constants [k sub ⁄ K m(D) ] for some substrates (i.e. linker cleavage probably shifts the substrate specificity profile of P-gp). Thus, this result also suggests that the linker region regulates the inherent substrate specificity of P-gp.Abbreviations

show abstract

Section: Discussionsupporting

confidence: 81%

Section: Protease Treatment Of Purified P-gp In Detergent Micellesmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Functional role of the linker region in purified human P‐glycoprotein

et al. 2009

View full text Add to dashboard Cite

show abstract

“…Tariquidar has been reported to act as a noncompetitive inhibitor of P-gp (25). Tariquidar may resemble modulators such as flupentixols that appear to modulate ATPase activity but bind outside of the drug-binding sites (60,61). Perhaps binding of tariquidar traps P-gp in an outward-facing conformation that mimics cross-linking (states IV-VI, Fig.…”

Section: Discussionmentioning

confidence: 99%

The ATPase Activity of the P-glycoprotein Drug Pump Is Highly Activated When the N-terminal and Central Regions of the Nucleotide-binding Domains Are Linked Closely Together

Loo

Bartlett

Detty

et al. 2012

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: P-glycoprotein is an ABC transporter that confers multidrug resistance. Results: Linkage of the nucleotide-binding domains with short but not long cross-linkers highly activated ATPase activity. Conclusion: Drug substrates activate P-gp ATPase activity by promoting close association of the nucleotide-binding domains. Significance: Understanding the mechanism of P-glycoprotein will aid in the development of inhibitors to overcome multidrug resistance.

show abstract

“…The authors concluded that modulation of P-gp function by cis-flupentixol (76) was mediated through interaction of modulator with P-gp at a site which was specific for tricyclic compounds containing thioxanthene or the phenothiazine backbone. Allosteric modulation of P-gp by flupentixols involved conformational changes which mimicked catalytic transition intermediates [181].…”

Section: Phenothiazines and Related Compounds As Modulators Of Mdr Trmentioning

confidence: 99%

The Role of the Membrane Actions of Phenothiazines and Flavonoids as Functional Modulators

Michalak

Wesołowska

Motohashi

et al.

Topics in Heterocyclic Chemistry

View full text Add to dashboard Cite

Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates

Cited by 11 publications

References 57 publications

Functional role of the linker region in purified human P‐glycoprotein

Functional role of the linker region in purified human P‐glycoprotein

The ATPase Activity of the P-glycoprotein Drug Pump Is Highly Activated When the N-terminal and Central Regions of the Nucleotide-binding Domains Are Linked Closely Together

The Role of the Membrane Actions of Phenothiazines and Flavonoids as Functional Modulators

Contact Info

Product

Resources

About