Affiliation:
1. Department of Biochemistry, Semmelweis University
2. HCEMM-SE Molecular Channelopathies Research Group
3. HUN-REN-SE Ion Channel Research Group
Abstract
CFTR, the anion channel mutated in cystic fibrosis patients, is a model ABC protein whose ATP-driven conformational cycle is observable at single-molecule level in patch-clamp recordings. Bursts of CFTR pore openings are coupled to tight dimerization of its two nucleotide-binding domains (NBDs) and in wild-type (WT) channels are mostly terminated by ATP hydrolysis. The slow rate of non-hydrolytic closure – which determines how tightly bursts and ATP hydrolysis are coupled – is unknown, as burst durations of catalytic site mutants span a range of ~200-fold. Here, we show that Walker A mutation K1250A, Walker B mutation D1370N, and catalytic glutamate mutations E1371S and E1371Q all completely disrupt ATP hydrolysis. True non-hydrolytic closing rate of WT CFTR approximates that of K1250A and E1371S. That rate is slowed ~15-fold in E1371Q by a non-native inter-NBD H-bond, and accelerated ~15-fold in D1370N. These findings uncover unique features of the NBD interface in human CFTR.
Funder
EU Horizon 2020 Research and Innovation Program
Cystic Fibrosis Foundation
National Research, Development and Innovation Office
Ministry for Innovation and Technology
Publisher
eLife Sciences Publications, Ltd
Subject
General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience