A topological switch in CFTR modulates channel activity and sensitivity to unfolding.

Jan Steyaert,Chloé Martens,Marie Overtus,Daniel Scholl,Yiting Wang,Cédric Govaerts,Rafael Colomer Martinez,Els Pardon,Abel Garcia-Pino,David N Sheppard,Jelle Hendrix,Maud Sigoillot,Toon Laeremans

doi:10.1038/s41589-021-00844-0

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.

Highlights

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR)anion channel that lead to impaired epithelial ion transport with dramatic consequences in multiple organs, the lungs[1,2]
We identify the molecular determinants of the equilibrium and show that the conformational transitions favour local unfolding of NBD1 in the presence of the F508del mutation, elucidating the conformational pathway involved in the pathogenesis of Cystic Fibrosis (CF)
Line, there is no conformational exchange during the passage of the protein through the confocal volume of the single molecule Förster resonance energy transfer (smFRET) microscope (~1–5 ms), indicating that the protein exists in one conformation at a time

Summary

Introduction

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR)anion channel that lead to impaired epithelial ion transport with dramatic consequences in multiple organs, the lungs[1,2]. Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). In most CF patients, the disease is caused by deletion of the phenylalanine residue at position 508 (F508del) in NBD1, which leads to absence of the channel from the plasma membrane[4]. Studies have shown that F508del compromises CFTR by decreasing the thermal stability of NBD1 and subsequently of the entire protein[6,9]. In vitro studies demonstrate that F508del has little effect on the NBD1 structure per se, but rather alters stability and dynamics of the domain[6,12,13]. We can gain molecular insight from stabilizing mutations that compensate for the effects of

Methods

Results

Conclusion