Dataset Information

Structural basis for the modulation of MRP2 activity by phosphorylation and drugs

ABSTRACT: Chemotherapy resistance by ABC transporters and secondary transporters is well established. Liver cancer is hard to treat by chemotherapy due to the presence of the multidrug resistance-associated protein 2 (MRP2/ABCC2) in addition to other transporters. The activity of MRP2 can be modulated by phosphorylation of its regulatory domain but also by drug-drug interactions. Here, we have determined the cryo-EM structure of MRP2 from Rattus norvegicus in an autoinhibited state (nucleotide-free) and in the presence of the drug modulator probenecid (uricosuric agent). The autoinhibited conformation is caused by the partially phosphorylated regulatory domain that folds within the transmembrane domain. Combining, in vitro phosphorylation of MRP2, mass spectrometry and kinetics, we show that full phosphorylation of the regulatory domain is required to relieve the autoinhibitory state due to steric clashes between the phosphate groups and the transmembrane domain. The drug bound state, has identified two drug binding sites that has allowed us to propose how probenecid modulates the activity of the transporter in addition to drug-drug interactions. Overall, we have structurally characterised the clinically relevant transporter MRP2, and we provide evidence on its modulation by intracellular kinases but also in the presence of external drugs. Finally, we mapped the mutations that cause Dubin–Johnson syndrome onto the structure and gain an understanding on their contribution in MRP2 dysfunction.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Graeme Benstead-Hume

LAB HEAD: Jyoti Choudhary

PROVIDER: PXD045845 | Pride | 2024-02-05

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 17

Similar Datasets

Project description:The GntR-like protein NorG has been shown to affect Staphylococcus aureus genes involved in the resistance to quinolones and beta-lactams such as those encoding the NorB and AbcA transporters. To identify the target genes regulated by NorG, we carried out transcriptional profiling assays using S. aureus RN6390 and its isogenic norG::cat mutant. Our data showed that NorG positively affected the transcription of global regulators mgrA, arlS, and sarZ. The three putative drug efflux pump genes most positively affected by NorG were the NorB efflux pump (5.1-fold), the MmpL-like protein SACOL2566 (5.2-fold), and the BcrA-like drug transporter SACOL2525 (5.7-fold). The S. aureus predicted MmpL protein showed 53% homology with the MmpL lipid transporter of Mycobacterium tuberculosis, and the putative SACOL2525 protein showed 87% homology with the bacitracin drug transporter BcrA of Staphylococcus hominis. Two pump genes most negatively affected by NorG were NorC (4-fold) and AbcA (6-fold). Other categories of genes such as those participating in amino acid, inorganic ion, or nucleotide transporters and metabolism, were also affected by NorG. Real-time RT-PCR assays for mgrA, arlS, sarZ, norB, norC, abcA, mmpL, and bcrA-like were carried out to verify microarray data and showed the same level of up- or down regulation by NorG. The norG mutant showed a twofold increase in the resistance to norfloxacin and rhodamine, both substrates of the NorC transporter, which is consistent with the resistance phenotype conferred by overexpression of norC on a plasmid. These data indicate that NorG has broad regulatory function in S. aureus. The goal of this study was to define the transcriptional response of S. aureus norG mutant cells. To do so, commercially available S. aureus Affymetrix GeneChips (Santa Clara, CA) were used to compare the expression properties of wild type (ISP794) and isogenic norG (QT11) mutant cells. S. aureus strains ISP794 and QT11 were grown to mid exponential phase growth, lysed , total bacterial RNA was extracted, labeled and hybridized to commerically available S. aureus Affymetrix GeneChips. In total three biological replicates were compared for each strain background.

			Action	DRS
	20230801_BLK03.raw	Raw
	20230801_BLK06.raw	Raw
	20230801_BLK09.raw	Raw
	20230801_BLK12.raw	Raw
	20230801_BLK15.raw	Raw

Dataset Information

Structural basis for the modulation of MRP2 activity by phosphorylation and drugs

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets