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, Tip W.; Bartlett, M. Claire; Detty, Michael R.; Clarke, David M.

doi:10.1074/jbc.m112.376202

Cited by 57 publications

(63 citation statements)

References 66 publications

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“…Mutations were introduced into the human P-gp cDNA as described previously (30). Mutants were expressed in HEK 293 cells for 18 h in the presence or absence of 10 M cyclosporine A.…”

Section: Methodsmentioning

confidence: 99%

“…The results may also explain the puzzling difference between maturation of NBD2 deletion mutants of P-gp and its sister protein, CFTR. P-gp and CFTR are both ABC proteins predicted to have similar structures (30). Deletion of NBD2 in P-gp inhibits P-gp maturation, whereas deletion of NBD2 in CFTR does not.…”

Section: Discussionmentioning

confidence: 99%

“…TMD1-NBD2 interactions may be particularly important for folding of P-gp into a native structure during synthesis because a ⌬NBD2 truncation mutant does not mature (28). By contrast, deletion of NBD2 in the CFTR protein, which has a similar structure to P-gp (30), yielded a mature product (28) that was as stable as its full-length counterpart (27).…”

Section: Mutations In Ih2 But Not Ih1 Inhibit Maturation Of P-gp-mentioning

confidence: 99%

“…3, A and B). Mutant T333C/L975C can be cross-linked when intact cells expressing the mutant are treated with BMOE cross-linker (30). Mutant T333C/L975C, however, does not show cross-linking if membranes containing the mutant are treated only with BMOE (Fig.…”

Section: Mutations In Ih2 But Not Ih1 Inhibit Maturation Of P-gp-mentioning

confidence: 99%

“…Extracellular Segments-Mutant T333C/L975C contains cysteines predicted to reside at the extracellular ends of TM segments 6 and 12, respectively (30). The double cysteine T333C/ L975C constructs (with or without the F1086A or A266F mutations) were transiently expressed in HEK 293 cells.…”

Section: Effect Of Mutations On Atp-dependent Cross-linking Of P-gpmentioning

confidence: 99%

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Human P-glycoprotein Contains a Greasy Ball-and-Socket Joint at the Second Transmission Interface

Loo

Bartlett

Clarke

2013

Journal of Biological Chemistry

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Background:The human P-glycoprotein drug pump confers multidrug resistance. Results: Hydrophobic suppressors in intracellular loop 2 restored activity to mutants with defective coupling between the ATPand drug-binding domains. Conclusion:A greasy "ball-and-socket" joint connects the ATP-and drug-binding domains. Significance: This work identifies key component of the drug pump mechanism and a potential target to modulate this clinically important protein.

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“…Mutations were introduced into the human P-gp cDNA as described previously (30). Mutants were expressed in HEK 293 cells for 18 h in the presence or absence of 10 M cyclosporine A.…”

Section: Methodsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Mutations In Ih2 But Not Ih1 Inhibit Maturation Of P-gp-mentioning

confidence: 99%

Section: Mutations In Ih2 But Not Ih1 Inhibit Maturation Of P-gp-mentioning

confidence: 99%

Section: Effect Of Mutations On Atp-dependent Cross-linking Of P-gpmentioning

confidence: 99%

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Human P-glycoprotein Contains a Greasy Ball-and-Socket Joint at the Second Transmission Interface

Loo

Bartlett

Clarke

2013

Journal of Biological Chemistry

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Novel Strategies Preventing Emergence of MDR in Breast Cancer

Malviya¹,

Singh²,

Yadav³

2023

Multi‐Drug Resistance in Cancer

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Protein Contacts and Ligand Binding in the Inward‐Facing Model of Human P‐Glycoprotein

2013

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The primary aim of this work was to analyze the contacts between residues in the nucleotide binding domains (NBDs) and at the interface between the transmembrane domains (TMDs) and the NBDs in the inward-open homology model of human P-glycoprotein (P-gp). The analysis revealed communication nets through hydrogen bonding in the NBD and at the NBD-TMD interface of each half involving residues from the adenosine triphosphate (ATP) motifs and the coupling helices of the intracellular loops. Similar networks have been identified in P-gp conformations generated by molecular dynamics simulation. Differences have been recorded in the networking between both halves of P-gp. Many of the residue contacts have also been observed in the X-ray crystal structures of other ATP binding cassette (ABC) transporters, which confirms their validity. Next, possible binding pockets involving residues of importance for the TMD-NBD communication were identified. By studying these pockets, binding sites were suggested for rhodamine 123 (R-site) and prazosin (regulatory site) at the NBD-TMD interface that agreed with the experimental data on their location. Additionally, one more R-site in the protein cavity was proposed, in accordance with the available biochemical data. Together with the previously suggested Hoechst 33342 site (H-site), all sites were interpreted with respect to their effects on the protein ATPase activity, in correspondence with the experimental observations. Several residues involved in key contacts in the P-gp NBDs were proposed for further targeted mutagenesis experiments.

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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

Cited by 57 publications

References 66 publications

Human P-glycoprotein Contains a Greasy Ball-and-Socket Joint at the Second Transmission Interface

Human P-glycoprotein Contains a Greasy Ball-and-Socket Joint at the Second Transmission Interface

Novel Strategies Preventing Emergence of MDR in Breast Cancer

Protein Contacts and Ligand Binding in the Inward‐Facing Model of Human P‐Glycoprotein

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