Structure of the Cystic Fibrosis transmembrane conductance regulator, what does this mean for future Cystic Fibrosis research?

By Bryony Ackroyd

Twitter: @BryonyAckroyd

In a previous blog post the implications of a mutated Cystic Fibrosis transmembrane conductance regulator (CFTR) receptor in Cystic Fibrosis (CF) was discussed, along with the pros and cons of the break through drug, Ivacaftor. Following on from this, in December 2016, the structure of the CFTR from zebrafish was determined via electron cryo-microscopy, how will this implicate future CF research?

CF is a genetic disease that affects 70,000 people worldwide and is characterised by an overly viscous mucus lining of the airways, resulting in difficulty in clearing the airways by coughing, and an increase in infections from opportunistic pathogens.  CF is caused by different mutations in the CFTR, an ABC transporter ion channel, which results in an imbalance in ion concentration and the observed phenotype of highly viscous mucus. The CFTR conducts chloride and as well as regulating other ion channels, such as chloride channels and glutathione transport. There are approximately 1900 known mutations within the CFTR, which is primarily expressed within the airway submucosal glands in the lungs.

Although the structure is of the zebrafish CFTR, the human and zebrafish CFTR share 55% sequence identity and 42 of the 46 mutations that cause CF are identical, making the zebrafish CFTR structure a useful tool for studying human CF.

The structure of the zebrafish CFTR is in the inwards facing conformation, i.e. open to the cytoplasm and closed to the outside of the cell. The electron microscopy (EM) density for the 12 transmembrane helices of the CFTR was good enough to unambiguously assign the amino acids. However, the density for the nucleotide binding domains (NBDs) was not as sharply resolved, therefore the crystal structures of the human and mouse NBDs were used as a way to guide model building of the zebrafish NBDs.

cftr-structure
Structure of the zebrafish CFTR, determined via electron cryo-microscopy. The R domain and related density is shown in yellow, the Lasso motif is shown in red, transmembrane domin 1 in blue and transmembrane domain 2 in green. The lasso domain is shown to be partially integrated into the membrane and in close proximity to the R domain.

 

When determining the structure of the CFTR it was found to contain an “N-terminal interfacial structure” which has never previously been seen in an ABC transporter, it is referred to as the lasso motif.  The first 40 resides of the lasso motif are within the membrane and pack against one of the transmembrane helices. The part of the lasso motif extending outside the membrane forms a helix and tucks under helix one of the CFTR. Many of the mutations causing CF are found within the lasso motif region, highlighting its importance in the disease. Some hypotheses have suggested that the lasso motif regulates channel gating through interactions with the R domain, which fits well with the symptoms of CF. The R domain of the CFTR appears to inhibit the channel in the dephosphorylated state, this inhibition is reversed when the R domain is phosphorylated.

The missense CF-causing mutations were then mapped onto the structure of the CFTR, making it possible to categorise the mutations into 4 groups, pore construction mutations, folding mutations, ATPase site mutations and NBD/Transmembrane domain interface mutations. Pore construction mutations include mutations expected to alter the structure or electrostatics of the pore. Mutations that destabilised the CFTR and therefore caused folding mutations were classified as folding mutations.  ATPase site mutations comprised of mutations within the NBDs that are thought to interfere with ATP binding and the formation of the closed NBD dimer. NBD/transmembrane domain mutations cause defects in folding and gating and therefore impact on the transmission of conformational changes from the NBDs to the transmembrane domains.

The determination of the structure of the zebrafish CFTR has been a much needed breakthrough within the CF research field. For the first time researchers have been able to accurately pinpoint mutations involved in CF, giving a much greater insight into how these mutations cause the observed symptoms and allowing rational drug design to target these problem points.  This advancement can only be a positive thing for the future CF research.

 

Source: Zhang, Zhe et al., (2016). Atomic Structure of the Cystic Fibrosis Transmembrane Conductance Regulator. Cell, Volume 167 , Issue 6 , 1586 – 1597.e9.

Ivacaftor, the Miracle Drug?

 

Ivacaftor has been hailed as a miracle drug for the treatment of Cystic fibrosis (CF). Although Ivacaftor is not designed for every CF sufferer, it is life changing for those who benefit from it.

CF is a genetic disease that affects 70,000 people worldwide and is characterised by an overly viscous mucus lining of the airways, resulting in difficulty in clearing the airways by coughing, and an increase in infections from opportunistic pathogens.  CF is caused by different mutations in the Cystic fibrosis transmembrane conductance regulator (CFTR), an ABC transporter ion channel, which results in an imbalance in ion concentration and the observed phenotype of highly viscous mucus. The CFTR conducts chloride and as well as regulating other ion channels, such as chloride channels and glutathione transport. There are approximately 1900 known mutations within the CFTR, which is primarily expressed within the airway submucosal glands in the lungs.

Until now all CF care has been supportive rather than curative. However a recent breakthrough with the drug Ivacaftor, which treats the underlying problem rather than just the symptoms of CF, could change the CF care landscape. Ivacaftor is used to treat CF patients with one of a set of specific mutations including G551D, which affects approximately 4% of CF patients and is the third most common mutation. The G551D mutation is characterised by correct positioning of the CFTR on the epithelial cell surface but incorrect function, the CFTR is unable to transport chloride ions.

Slide1
Predicted structure of the CFTR. TMD; Transmembrane Domain, NBD; Nucleotide Binding Domain. The G551D mutation occurs in the NBD2 region and prevents ATP dependent gating. Adapted from Kim and Skach, 2012 (http://dx.doi.org/10.3389/fphar.2012.00201).

Ivacaftor, developed by Vertex Pharmaceuticals together with the Cystic fibrosis Foundation, assists with the function of the mutant CFTR by directly binding the CFTR channel. Binding increases the probability of channel opening by inducing opening of the ion channel independent of ATP binding and hydrolysis. This reduces the imbalance in the ion concentration and lowers the viscosity of the mucus in the airways allowing easier breathing of the CF patient. CF sufferers who have had access to Ivacaftor have been able to “run without coughing” and “take deep breaths”.

The FDA approved Ivacaftor in January 2012 and the combination drug with lumacaftor gained FDA approval in July 2015. The inclusion of lumacaftor in the combination drug enables treatment of CF patients with a slightly different mutation in addition to those treated by Ivacaftor.

However there is a catch, Ivacaftor treatment costs $300,000 per patient per year. Patients are likely to be taking Ivacaftor for the rest of their lives and so the cost becomes astronomical. On the other hand Vertex has stated that they will make the drug available free of charge for those patients in the US without medical insurance and with a household income of less than $150,000 a year.

The real question surrounding Ivacaftor is; how to pay for the miracle?

 

Source: Kotha and Clancy (2013). Ivacaftor treatment of cystic fibrosis patients with the G551D mutation: a review of the evidence. Therapeutic Advances in Respiratory Disease, 7(5)288-296.

Bryony Ackroyd

Twitter: @BryonyAckroyd