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.

Essential trypanosome transporters hint at new therapeutic targets

By Rebecca Hall

Twitter: @RebeccaJHall13

Blog: http://www.ananxiousscientist.wordpress.com

 

Human African trypanosomiasis (HAT), more commonly known as sleeping sickness, is a disease caused by the protozoan parasite Trypanosoma. Endemic in 36 sub-Saharan African countries, HAT causes fever, headaches, joint pain and, once the parasite has crossed the blood-brain barrier, the characteristic sleep cycle disturbances that give the condition its colloquial name. Trypanosomes have a complex life cycle, residing partly inside its tsetse fly host and infecting mammals in a separate stage. The adaptations that the parasite has undergone in order to thrive inside humans enable it to evade the immune system; by ‘putting on’ a unique ‘coat’ of glycoproteins, trypanosomes ensure that the immune cells cannot keep up with its disguises. As such, developing drugs to combat sleeping sickness and nagana, its equivalent in cattle, is a complex and frequently unsuccessful process.

The drugs that are available currently to treat HAT are limited by a risk of toxicity and are not always effective. There is also increasing concern that resistance may arise and so there is a lot of interest in teasing out the biology of trypanosomes, with transport and metabolism being one key area. The hope is that they may be able to find new therapeutic targets by identifying essential components of the parasite.

Amino acid uptake is hugely important for trypanosomes. When they transition from mammalian to insect host they are required to adapt to very different environments. Blood is the sole diet of the tsetse and therefore the parasite must be able to survive on amino acids as their energy source when they are in this stage of their life cycle. They are also auxotrophic for a number of amino acids, meaning they cannot produce them themselves and instead rely on importing them to survive. The transporters for these therefore provide a potential drug target; block the ability to uptake essential metabolites and the parasite will die.

A paper published in early January describes two transporters that could become potential therapeutic targets. Mathieu et al. looked at two amino acids, arginine and lysine, that are essential for trypanosome survival. They identified candidate transporters by constructing a phylogenetic tree and transformed them into Saccharomyces cerevisiae mutants. These mutants were unable to uptake different amino acids and so the group were able to establish what these proteins transported by assessing the ability of the mutants to grow on various substrates. They identified transporters that enabled growth on lysine and arginine in strains of S. cerevisiae that would otherwise have been unable to grow.

The team then used transport assays to reveal that these transporters have both high affinity and selectivity for their substrates. Transcriptomics suggested that they are highly expressed and analysis of cMyc-tagged trypanosomes indicated that these transporters localise in the plasma membrane. They finally assessed the essentiality of these proteins by down-regulating their expression through RNA interference and found that growth of these parasites was significantly reduced.

These transporters are therefore interesting therapeutic candidates because of the reliance of the trypanosome on them for survival. Importantly, these are not related to uptake systems in humans and so any drug that worked against them would not run the risk of off-target effects.

 

Source: Arginine and lysine transporters are essential for Trypanosoma brucei, Mathieu et al. (2017), PLOS ONE

 

 

Sequence-independent assay for importers results in validation of novel thiamine uptake system

By Ivan Gyulev

Twitter: @IvanGyulev

A study published in October 2016 in Nature Chemical Biology by Prof Morten Sommer and colleagues reported the use of a sequence-independent screen for the identification of novel bacterial small molecule transporters. The assay is based on a synthetic selection system that relies on riboswitch biosensors. A riboswitch (small molecule-binding RNAs) is located in the 5’UTR of an antibiotic resistance gene and inhibits its translation by sequestering its ribosome binding sites. However, when the riboswitch’s ligand is present in sufficient concentration intracellularly, the translational repression is alleviated and the gene is expressed, thereby conferring resistance against its respective antibiotic. By using two antibiotics and two resistance genes, the researchers dramatically reduced the rate of false positive mutants. Using this assay, one can screen a library of metagenomic fragments for ligand importers. To screen for importers of a new ligand researchers only need to change the riboswitch. Genee et al. demonstrated the modularity of their design by implementing it in the discovery of thiamine and xanthine importers.

The outline of the synthetic selection system in the case of selection for thiamine importers (using the ThiM19 riboswitch) is shown below (taken from Figure 1a from the paper).

slide1
Figure 1. Synthetic selection system for thiamine uptake. (a) The dual ribosome binding site (RB S) selection system controlling chloramphenicol-resistance and spectinomycin-resistance genes (cat and aadA). Translation of the resistance genes is enabled only after binding of TPP. The dual selection reduces the number of false positives, as false triggering (e.g., by mutation of one riboswitch) will not lead to cell growth.

After validating the synthetic selection system the authors then screened metagenomic DNA libraries from soil and gut fecal samples for thiamine importers and discovered a novel class of thiamine importer – PnuT (screen strategy outlined in Figure 2a from the paper).

slide1
Figure 2. Functional metagenomic selection of thiamine transporter. (a) Total DNA extracted from soil and gut fecal samples (metagenomic DNA) was fragmented into ~2-kb fragments, cloned into an expression vector and transformed into an E. coli host strain harboring the thiamine selection system. The cell library was plated on selective growth medium supplemented with low amounts of thiamine. Cells that expressed a thiamine-uptake transporter from the metagenomic DNA insert imported extracellular thiamine and had increased intracellular TPP concentrations, leading to induction of riboswitch-mediated antibiotic resistance.

PnuT has homology to the nicotinamide riboside and nicotinamide mononucleotide transporter PnuC and had been previously predicted to be involved in thiamine uptake. PnuT’s function as a thiamine transporter was validated by selective growth and intracellular thiamine quantification by HPLC. Further bioinformatics analysis, revealed that PnuT is very common in the Bacteroidetes phylum. The authors then looked at phylogeny and the pattern of Pnu transporters’ co-localization with genes from thiamine salvage or biosynthesis pathway across genomes.

Finally, a previously published synthetic riboswitch (derived from aptamer identified by SELEX) was utilized to select for xanthine importers. The screen resulted in the isolation of two unique ORFs with more than 99% sequence identity at the amino acid level with known xanthine permeases from the NAT/NCS2 nucleobase-ascorbate transporter family. In both screens, fragments containing multi-drug resistance proteins were isolated

The authors highlight several limitations of the current screen technique –firstly, the discovery of transporters relying on multiple protein complexes (such as the thiamine importer from Bacillus/Clostridium ECF-ThiT or the E.coli ThiBPQ) would require larger metagenomic (or genomic) fragments (in the present study the range was between 1kb and 3kb but it is possible to use larger fragments). Secondly, these proteins are not necessarily encoded in the same chromosomal region. Thirdly, naturally-occurring riboswitches and allosteric transcription factors are the go-to choice for small-molecule biosensors but synthetic riboswitches are more difficult to develop synthetically. Reportedly, one way to go around this is to construct a metabolic pathway bridging an undetectable compound to a detectable one.

Altogether, the novel synthetic selection strategy is a powerful tool for the isolation and validation of novel importers from metagenomic libraries or putative transporters from genomic sequences. It is also impressive that in its first implementation the assay led to the experimental validation of a novel import system.

 

Source: Genee, H.J. et al., (2016).Functional mining of transporters using synthetic selections. Nat. Chem. Biol.  12, 1015-1022.