Small-angle X-ray scattering study of the ATP modulation of the structural features of the nucleotide binding domains of the CFTR in solution

Abstract

Nucleotide binding domains (NBD1 and NBD2) of the cystic fibrosis transmembrane conductance (CFTR), the defective protein in cystic fibrosis, are responsible for controlling the gating of the chloride channel and are the putative binding site for several candidate drugs in the disease treatment. We studied the structural properties of recombinant NBD1, NBD2, and an equimolar NBD1/NBD2 mixture in solution by small-angle X-ray scattering. We demonstrated that NBD1 or NBD2 alone have an overall structure similar to that observed for crystals. Application of 2 mM ATP induces a dimerization of NBD1 but does not modify the NBD2 monomeric conformation. An equimolar mixture of NBD1/NBD2 in solution shows a dimeric conformation, and the application of ATP to the solution causes a conformational change in the NBD1/NBD2 complex into a tight heterodimer. We hypothesize that a similar conformation change occurs in situ and that transition is part of the gating mechanism. To our knowledge, this is the first direct observation of a conformational change of the NBD1/NBD2 interaction by ATP. This information may be useful to understand the physiopathology of cystic fibrosis.

A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease

“…P31–43 (but not P57–68) bound to rhNBD1 (Fig G), and this interaction was attenuated for both rhNBD1 mutants (Fig H). A higher molecular weight complex of biotinylated P31–43 bound to NBD1 was also detectable, in line with previous reports of higher molecular weight aggregates of NBD1 occurring in the presence of 2 mM ATP (Galeno et al , ; Fig G and H). To confirm the hypothesis that NBD1 interacts with P31–43, we replaced endogenous CFTR with mutant versions bearing either F400A/E403A or P439A/P477A double NBD1 mutations in Caco‐2 cells (that were first rendered CFTR‐null by CRISP/CAS9 technology and then transfected with mutant CFTR or WT‐CFTR as a control).…”

“…CF is best known for its respiratory phenotype, yet also frequently leads to intestinal problems, as CFTR protein is strongly expressed all along the intestine (Gadsby et al , ; Ooi & Durie, ). CFTR is not only an anion channel, but also orchestrates proteostasis at respiratory and intestinal epithelial surfaces, meaning that it regulates adaptation to cell‐autonomous or environmental stress signals (Luciani et al , ; Villella et al , ,b; Ferrari et al , ). CFTR malfunction generates epithelial stress, early TG2 activation, inhibition of autophagy, and activation of innate immunity (Maiuri et al , ; Luciani et al , ; Luciani et al , ; Villella et al , ,b; Ferrari et al , ), features that are reminiscent of those triggered by gliadin in intestinal epithelial cells and celiac duodenal mucosa (Maiuri et al , ; Meresse et al , ; Barone et al , ).…”

On the interactions between nucleotide binding domains and membrane spanning domains in cystic fibrosis transmembrane regulator: A molecular dynamic study

“…The CFTR model used in the simulations is reputed to correspond to the open channel conformation, and therefore there is a close interaction between the NBDs [39e41], in the head-to-tail conformation [7,12,13,42,43]. We have identified the interacting residues of the NBDs, either forming hydrogen bonds, or presenting atom pairs in close contact at a distance 3.0 Å.…”

“…Also, structural changes of the whole CFTR and recombinant RD upon phosphorylation have been studied by cryoelectron microscopy [3,4], and small-angle x-ray scattering (SAXS) [5]. Furthermore, this latter scattering technique was also proficient in the observation of NBDs conformational changes associated to ATP-binding [6,7]. Nevertheless, the only structural data available at atomic resolution of CFTR come from recombinant NBDs [8e10].…”