Összes szerző

Szakács Gergely

az alábbi absztraktok szerzői között szerepel:

Goda Katalin
Crosstalk between nucleotide and substrate binding in ABCG2

Aug 30 - szerda

09:00 – 09:20

Membránok és membránfehérjék biofizikája

E21

Crosstalk between nucleotide and substrate binding in ABCG2

Zsuzsanna Gyöngy1,2, Gábor Mocsár1, Zsuzsanna Ritter1,2, Thomas Stockner3, Gergely Szakács4,5, Katalin Goda1

1 Department of Biophysics, Faculty of Medicine, University of Debrecen, Hungary

2 Doctoral School of Molecular Cell and Immune Biology, University of Debrecen

3 Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Austria

4 Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary

5 Institute of Cancer Research, Medical University of Vienna, Austria

ABCG2 is an exporter type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells and tumor stem cells, it may cause multidrug resistance contributing to the failure of chemotherapy. A better understanding of the molecular mechanism of ABCG2 may provide new therapeutic targets to improve the treatment of drug resistant tumors. In the present work, we designed fluorescence-based assays to investigate the affinity of ABCG2 to transported drugs and nucleotides in live or semi-permeabilized cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the 5D3-reactive inward-facing (IF) to a 5D3-dim outward-facing (OF) conformation is induced by nucleotide binding, and this conformational transition is accelerated by substrates and hindered by the known inhibitor Ko143. The drop of 5D3 binding occurred simultaneously with the decrease of substrate binding, suggesting that the high-to-low switch in drug binding affinity might coincide with the transition from the IF to the OF conformation. Low substrate binding persists in the vanadate-trapped post-hydrolysis state, indicating that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

Acknowledgment

We are grateful for the financial supports by the Hungarian National Research, Development and Innovations Office (NKFIH; https://nkfih.gov.hu/english), grant number K124815 and GINOP-2.2.1-15-2017-00079. GS was supported by a Momentum Grant of the Hungarian Academy of Sciences.

Szikszainé Ritter Zsuzsanna
Analysis of unilateral Walker A and A-loop mutants indicate that a single active catalytic site is sufficient to promote transport in ABCB1

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P55

Analysis of unilateral Walker A and A-loop mutants indicate that a single active catalytic site is sufficient to promote transport in ABCB1

Zsuzsanna Ritter1,2, Szabolcs Tarapcsák1, Zsuzsanna Gyöngy1,2, Orsolya Bársony1, Nimrah Ghaffar1,2 Thomas Stockner3, Gergely Szakács4, and Katalin Goda1

1Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
2Doctoral School of Molecular Cell and Immune Biology, University of Debrecen, Debrecen, Hungary
3Institute of Pharmacology, Center for Physiology and Pharmacology,
4Institute of Cancer Research, Medical University of Vienna, Vienna, Austria

The human ABCB1 is a full transporter with two nucleotide binding domains (NBDs) and two pseudo-symmetric transmembrane domains (TMDs). The two NBDs form two symmetrically arranged composite nucleotide binding sites (NBSs). Each NBS is formed by the A-loop, H-loop, Walker A, Walker B and Q-loop of one NBD, and the X-loop and signature sequence of the other NBD. The conserved tyrosine of the A-loop aligns the adenine ring of the bound ATP, contributing to nucleotide binding affinity through stacking interactions. The Walker A lysine interacts with the α and β phosphate of ATP. The two nucleotide binding domains were shown to be functionally equivalent, and the integrity of both catalytic centers is generally believed to be needed for transport. Consistently with the widely accepted models predicting that the two NBDs hydrolyze ATP in a strictly alternating order, unilateral mutation of these residues have been described to disrupt ATP hydrolysis and even affect ATP binding.

Here we demonstrate that while ABCB1 variants carrying bilateral A-loop or Walker A mutations are completely inactive, the unilateral exchange of the A-loop tyrosine to alanine or the unilateral mutation of the Walker A lysine to methionine is compatible with both ATP hydrolytic activity and transport function. Characterization of the single mutants revealed the significant (about 10-fold) reduction of the apparent ATP binding affinity compared to wild-type ABCB1. Stabilization of the post-hydrolytic complex by phosphate mimicking anions, such as vanadate or BeFx also occurred at higher ATP concentrations compared to wild-type, supporting that the mutated site probably has an effect on the overall conformation of the NBD dimer. Although the basal catalytic activity was strongly reduced in accordance with the decreased ATP binding affinity of the single mutants, the degree of ATPase stimulation by verapamil was almost identical to that of the wild-type, showing that drug-stimulation of the ATPase activity is preserved in the single mutants. Location of the mutation in the N or C terminal NBD did not affect the extent of ATPase stimulation by verapamil. Taken together, our data indicate that, in contrast to prevailing views, single-site NBD mutant ABCB1