Conformational Variability in Ground-State CFTR Lipoprotein Particle Cryo-EM Ensembles.

Cystic fibrosis transmembrane regulator (CFTR) is a dynamic membrane protein belonging to the ABC transporter family. It is unusual within this family as it is an ion channel, as opposed to a transporter. Activation of CFTR requires ATP and phosphorylation by PKA, and dysregulation of CFTR mediated salt and water homeostasis can lead to cystic fibrosis.

Cryo-EM Visualization of an Active High Open Probability CFTR Anion Channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, crucial to epithelial salt and water homeostasis, and defective due to mutations in its gene in patients with cystic fibrosis, is a unique member of the large family of ATP-binding cassette transport proteins. Regulation of CFTR channel activity is stringently controlled by phosphorylation and nucleotide binding. Structural changes that underlie transitions between active and inactive functional states are not yet fully understood.

R-Domain Phosphorylation by Protein Kinase A Stimulates Dissociation of Unhydrolyzed ATP from the First Nucleotide-Binding Site of the Cystic Fibrosis Transmembrane Conductance Regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an asymmetric ATP-binding cassette transporter in which ATP hydrolysis occurs only at the second of the two composite nucleotide-binding sites whereas there are noncanonical substitutions of key catalytic residues in the first site. Therefore, in widely accepted models of CFTR function, ATP is depicted as remaining bound at the first site while it is hydrolyzed at the second site. However, the long lifetime of ATP at nucleotide-binding domain 1 (NBD1) had been measured under conditions where the channel had not been activated by phosphorylation.

The cubicon method for concentrating membrane proteins in the cubic mesophase.

The lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible-using traditional methods-to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. The cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution.

Thermal stability of purified and reconstituted CFTR in a locked open channel conformation.

CFTR is unique among ABC transporters as the only one functioning as an ion channel and from a human health perspective because mutations in its gene cause cystic fibrosis. Although considerable advances have been made towards understanding CFTR's mechanism of action and the impact of mutations, the lack of a high-resolution 3D structure has hindered progress. The large multi-domain membrane glycoprotein is normally present at low copy number and when over expressed at high levels it aggregates strongly, limiting the production of stable mono-disperse preparations.

Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains.

Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR.

Allosteric modulation balances thermodynamic stability and restores function of ΔF508 CFTR.

Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR (cystic fibrosis transmembrane conductance regulator) that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remains incomplete. Here, we show that the thermal instability of human ΔF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species.

The cystic fibrosis transmembrane conductance regulator (CFTR): three-dimensional structure and localization of a channel gate.

Cystic fibrosis affects about 1 in 2500 live births and involves loss of transmembrane chloride flux due to a lack of a membrane protein channel termed the cystic fibrosis transmembrane conductance regulator (CFTR). We have studied CFTR structure by electron crystallography. The data were compared with existing structures of other ATP-binding cassette transporters.

Domain location within the cystic fibrosis transmembrane conductance regulator protein investigated by electron microscopy and gold labelling.

The domain organisation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was studied using electron microscopy of detergent-solubilised dimeric complexes. Ni-NTA nanogold labelling data suggest that in the nonphosphorylated, nucleotide-free state, the C-terminus is intimately associated with the cytoplasmic ATP-binding regions, whilst part of the regulatory domain occupies a position close to the cytoplasmic surface of the lipid membrane. Removal of the entire second nucleotide binding domain (NBD2) results in a deficit in the CFTR structure that is consistent with the size and shape of a single NBD.

Regulatory insertion removal restores maturation, stability and function of DeltaF508 CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multidomain membrane protein that matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide-binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed.

Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR.

Deletion of PHE508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of CFTR, which causes most cystic fibrosis, disrupts the folding and assembly of the protein. Although the folding pathways and yield of isolated NBD1 are altered, its global structure is not, and details of the changes in the rest of the protein remain unclear. To gain further insight into how the whole mutant protein is altered, we have determined the influence of known second-site suppressor mutations in NBD1 on the conformation of this domain and key interfaces between domains.

Architecture of the cystic fibrosis transmembrane conductance regulator protein and structural changes associated with phosphorylation and nucleotide binding.

We describe biochemical and structural studies of the isolated cystic fibrosis transmembrane conductance regulator (CFTR) protein. Using electron cryomicroscopy, low resolution three-dimensional structures have been obtained for the non-phosphorylated protein in the absence of nucleotide and for the phosphorylated protein with ATP. In the latter state, the cytosolic nucleotide-binding domains move closer together, forming a more compact packing arrangement.

Multiple membrane-cytoplasmic domain contacts in the cystic fibrosis transmembrane conductance regulator (CFTR) mediate regulation of channel gating.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) ion channel mutated in patients with cystic fibrosis. The most common mutation, deletion of phenylalanine 508 (DeltaF508) and many other disease-associated mutations occur in the nucleotide binding domains (NBD) and the cytoplasmic loops (CL) of the membrane-spanning domains (MSD). A recently constructed computational model of the CFTR three-dimensional structure, supported by experimental data (Serohijos, A.

CFTR (ABCC7) is a hydrolyzable-ligand-gated channel.

As the product of the gene mutated in cystic fibrosis, the most common genetic disease of Caucasians, CFTR is an atypical ABC protein. From an evolutionary perspective, it is apparently a relatively young member of the ABC family, present only in metazoans where it plays a critical role in epithelial salt and fluid homeostasis. Functionally, the membrane translocation process it mediates, the passive bidirectional diffusion of small inorganic anions, is simpler than the vectorial transport of larger more complex substrates ("allocrites") by most ABC transporters.

Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR).

The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that is mutated in patients suffering from cystic fibrosis. Here we report the purification and first crystallization of wild-type human CFTR. Functional characterization of the material showed it to be highly active.