Összes szerző

Ghaffar Nimrah

az alábbi absztraktok szerzői között szerepel:

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