Efficient protein production inspired by how spiders make silk

By: Caroline Pearson

Twitter: @CarolineRosePea

Membrane proteins are notoriously difficult to produce recombinantly due to their hydrophobicity but a research team from Karolinska Institutet in Sweden have taken inspiration from nature to help overcome this difficulty. Spider silk is composed of large aggregation prone proteins called spidroins. Despite their aggregation prone nature these proteins can be produced at high concentrations without aggregation, before being passed through a narrow duct and converted into spider silk. The amphiphilic nature of spidroins with hydrophilic N- and C-terminal domains flanking hydrophobic centres allows them to arrange into micellar structures with hydrophilic terminal domains sequestering the hydrophobic and aggregation prone regions inside the micelle.

Previous research found that the hydrophilic N-terminal domain (NT) was highly conserved and mediates solubility of spidroins proteins above pH 6.5. However, as the proteins move towards the spinning duct the surrounding pH is decreased and NT form into antiparallel bundles through dipolar interactions which interconnect the proteins, allowing them to be spun into silk. In order to use spiroidins NT domain as a pH insensitive solubility tag, the research team produced a charge-reversed NT with Asp40 and Lys65 swapped in their positions to prevent antiparallel bundle formation. The novel NT mutant (named NT*) was shown to persist as a monomer over a wide pH range through size exclusion chromatography, and NMR spectroscopy revealed that the structure of the NT* was comparable to that of monomeric wild type NT. Promisingly this NT* also showed improved solubility, stability and refolding capacity.

The team then investigated whether NT* could enhance heterologous production of pharmaceutically relevant proteins that had previously been difficult to produce due to their high aggregation tendency. One such protein, SP-C, is an important surfactant protein that allows lungs to re-inflate after expiration of breath. It is also used to prevent respiratory distress in premature infants with insufficient amounts of lung surfactants. It is “perhaps the most hydrophobic peptide isolated from mammals”. Currently, surfactant preparations have to be extracted from animal lungs as recombinant production has proven difficult due to its hydrophobic nature.

N-terminal fusion with wild type NT and NT* enhanced recombinant production of an SP-C analogue compared to other fusion peptides used for comparison. Specifically, NT* resulted in production of mainly soluble fusion protein whereas wild type NT fusion produced mainly insoluble SP-C analogue. These results, along with successful production of a range of other pharmaceutically relevant peptides, suggest NT* as a novel solubility enhancing fusion tag with the potential to allow production of a wide variety of peptides that have previously been refractory to recombinant production. With ~60% of currently available pharmaceutical drugs targeting membrane associated proteins this is a positive step towards improved membrane protein research in drug development.

 

 

Source: Nina Kronqvist, Médoune Sarr, Anton Lindqvist, Kerstin Nordling, Martins Otikovs, Luca Venturi, Barbara Pioselli, Pasi Purhonen, Michael Landreh, Henrik Biverstål, Zigmantas Toleikis, Lisa Sjöberg, Carol V. Robinson, Nicola Pelizzi, Hans Jörnvall, Hans Hebert, Kristaps Jaudzems, Tore Curstedt, Anna Rising, Jan Johansson. Efficient protein production inspired by how spiders make silk. Nature Communications, 2017; 8: 15504

Lipid Trafficking: How diverse architectures of lipid transport systems shuttle lipids to the bacterial outer membrane.

By: Michelle Rudden

Twitter: @RuddenMichelle

 

Highly conserved domains in MCE proteins are used as structural building block to synthesise diverse transport architectures capable of shuttling hydrophobic lipid molecules between the inner and outer membrane of bacteria.

 

One of the defining hallmarks of Gram negative bacteria is their cell wall, composed of two phospholipid (PL) bilayer membranes separated by the periplasmic space which contains a thin layer of peptidoglycan. The inner membrane (IM) faces the cytoplasm while the outer membrane (OM) is exposed to the environment. The physical properties of the OM is a crucial selective barrier often preventing entry of toxic compounds, antibiotics and host innate immune compounds such as cationic antimicrobial peptides (CAMPs). Any mutations or changes affecting the physical integrity of the OM can reduce pathogenicity and increase antibiotic susceptibly, as a result it is a therapeutic target for many bacterial diseases. The OM is an asymmetric bilayer composed of an inner leaflet of phospholipids and an outer leaflet rich in lipopolysaccharide (LPS) in which several non-specific porins and specific uptake channels are embedded. The LPS-bilayer structure of the OM is more rigid compared to normal phospholipid bilayers and is largely impermeable to hydrophobic molecules by slowing down passive diffusion and narrow pores limit by size the uptake of hydrophilic molecules. The transport system for trafficking LPS molecules from the cytoplasm has been described in detail with a model comprising seven essential proteins (with most of the structures resolved) that spans the periplasmic space to deliver the LPS molecule to the outer leaflet of the OM. However there is no detailed mechanism described for the transport of nascent phospholipids in the inner leaflet and exactly how the asymmetry is maintained between the leaflets of the OM still remains unclear.

In a paper published this month in Cell, Ekiert et al, (2017) propose a mechanism by which proteins belonging to the mammalian cell entry (MCE) superfamily form hexameric assemblies that can form diverse protein channels with hydrophobic pores capable of shuttling hydrophobic molecules between the IM and OM in E. coli.  MCE proteins are ubiquitous in double membraned bacteria including E. coli and some membrane bound organelles such as chloroplasts where they are suggested to be involved in transport of lipid molecules either from the OM to the IM or as exporters driving molecules out of the cell.

Lipid molecules are hydrophobic and are poorly soluble in water, the hydrophilic periplasmic space is the major barrier in lipid transport across the membranes. In the LPS transport model protein components assemble to form a periplasm spanning bridge with a hydrophobic core that protects the lipid tails of LPS facilitating the movement of LPS molecules toward the OM. This led Ekiert et al (2017) to propose a similar mechanism by which MCE proteins assemble to form a hydrophobic pore allowing the transport of phospholipids between inner and outer membranes.

In this paper X-Ray and Cryo-electron microscopy (Cryo-EM) structures are reported for three MCE proteins found in E. coli each producing a distinct architecture with conserved MCE hexameric ring modules. The crystal structure of the MCE protein MlaD in E. coli revealed a single homo-hexameric ring assembled with a hydrophobic core that facilitates the transport of phospholipids. MlaD forms a complex with three other proteins anchored in the IM (Fig 1). MlaD does not span the periplasmic space, it recruits periplasmic lipid binding protein MlaC that delivers the phospholipids to the outer membrane.

Cryo EM structures of two different MCE proteins non-homologous to MlaD revealed different architectures that span the periplasm implicating their role in moving hydrophobic substrates across the membrane. PqiB forms a needle and syringe like structure that forms a periplasmic bridge (Fig 1). PqiB contains three MCE domains (MCE1, MCE2, MCE3) each forming a hexameric ring stacked on top of each other to form the hollow barrel of the structure (Fig. 1). The needle like structure of PqiB is a six helix coiled coil, one helix contributed from the C-terminus of each polypeptide chain. The needle contains a hollow hydrophobic lumen that runs through the barrel thus allowing for the movement of lipid molecules across the periplasm.  The YebT MCE protein also forms a periplasmic bridge with a unique structure. YebT contains seven MCE domains that stack on top of each other as hexameric rings forming an elongated tube,  like both MlaD and PqiB, YebT is anchored in the IM by N-terminal trans membrane (TM) helices.  The elongated tube also contains a hydrophobic lumen which allow the transport of hydrophobic substrates. Both YebT and PqiB are greater than 200 Å in length which is approximately the width of the periplasm. Ekiert et al., (2017) propose that YebT and PqiB provide a continuous pathway for lipid translocation across membranes without the need for a soluble periplasmic binding protein in a manner similar to that of the current LPS model.

Figure 1. Model for MCE-Mediated Phospholipid Transport across the Periplasm in E. coli
Figure 1. Model for MCE-Mediated Phospholipid Transport across the Periplasm in E. coli. Left, model for the Mla lipid transport system. MlaC likely serves as a soluble lipid carrier protein to transport lipids between the OM MlaA-OmpC/OmpF complex and the IM MlaFEDB complex. Middle left, PqiB may interact with OM lipoprotein PqiC and IM protein PqiA to mediate periplasmic lipid transport. Middle right, YebT may associate with YebS in the IM as part of a larger transport complex. Right, model of LPS transport system based on other studies (Okuda et al., 2016), which has also been suggested to form a bridge for LPS transport across the periplasm using a structurally unrelated framework.

Ekiert and colleagues’ present a neat structural argument for role of MCE domains in generating diverse architectures that are capable of spanning the periplasm and may function to traffic lipid across membranes. However the authors do state that there is still “a great deal that we don’t understand about the components of MCE transporters and how they function together”. This leads to several exciting open questions like do these systems function together or alone and are all three needed in E. coli? What is the directionality of lipid transport? What are the substrates for these systems? How phospholipids are transported and inserted in the outer membrane could be essential to understand how assembly and asymmetry is maintained in the OM.

 

 

Source: Ekiert et al., (2017) Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Cell 169, 273–285

Rapid evolution in transporters, and other membrane proteins, across the tree of life

By: Pejvak Moghimi

Twitter: @pezhvuk

I think all of us often indulge in metaphorical thinking when engaged with scientific concepts not directly visible to the naked eye, or tangible, to us. Transporters are no exception. Indeed, many of us, perhaps like to think of transporters in a similar way to the gateways that once regulated transport and commerce through the beautiful city walls of York. However, metaphors in science can quickly fall apart. And gateway metaphors are no exception. Transporters, unlike city gates of York are highly evolvable. That is not in itself ground-breaking, as all life, and the genes involved in regulating it, are subject to variety of evolutionary forces. However, Nick Lane and Victor Sojo have shown in a recent paper that membrane proteins (MPs), including transporters, undergo extraordinary rate of evolutionary changes in comparison to water-soluble proteins.

This study starts by investigating the number of orthologs of MPs and water-soluble proteins (WSPs), defined by the OMA database, among 64 species of Gram-positive and Gram-negative bacteria, Archaea, unicellular and multicellular eukaryotes. They compared the number of genes in orthologous groups (OGs) of MPs and WSPs and found that there are significantly smaller number of OGs in MPs than there are for WSPs for every species. Furthermore, they performed a logistic regression on the entire OMA ortholog data set and found that the probability of a gene coding a MP falls dramatically with increasing number of clades sharing that gene. They hypothesised that either;

  1. The sequence divergence is taking place at such a rapid rate that sequence-searching algorithms (such as the one used by BLAST) do not detect the more “hard-to-find” homologs, or
  2. It is due to disproportionate amount of gene loss in MPs.

Given the fact that OMA database uses a more rigorous version of the Smith-Waterman algorithm than BLAST, using OMA is a testament to the validity of the approach taken by the researchers to identify orthologs.

They followed the study by identifying which one of the evolutionary explanations satisfies their observations. To investigate whether rapid sequence divergence in MPs is involved in their observations, they used Nei’s sequence-diversity measure for 228,148 OMA OGs shared by any three or more species. They performed Welch’s t-test on the results and showed that MPs do indeed diverge more rapidly than WSPs. Interestingly, same analysis conducted on the same sequences, when dissected into exterior, transmembrane and interior-facing regions showed that the exterior-facing regions are under more rapid divergence rate than the transmembrane and interior-facing regions.

To determine that true gene loss, which is not to be confounded by an apparent gene loss caused by loss of homology beyond recognition, occurs at higher rate in MPs vs. WSPs they analysed prokaryotic clades (present in OMA), with 10 or greater number of closely related strains present in the database. They assumed that proteins shared by more than half of the number of clades are ancestral, and that clades, which do not share ancestral genes, represent true gene losses. The implicit assumption with this approach is that closely related strains are unlikely to have OGs that have diverged beyond recognition.

These analyses identifies that both rapid divergence and disproportionate gene loss are at play as parts of the molecular evolutionary dynamics of MPs. Authors report that previous research (citations included in the paper) demonstrates exported WSPs evolve faster than cytosolic proteins. This, together with the observations made in their work, led them to hypothesising a fundamental evolutionary principle:

“Membrane proteins evolve faster due to stronger adaptive selection in changing environments, whereas cytosolic proteins are under more stringent purifying selection in the homeostatic interior of the cell. This effect should be strongest in prokaryotes, weaker in unicellular eukaryotes (with intracellular membranes), and weakest in multicellular eukaryotes (with extracellular homeostasis).”

Two-fold effect of adaptation causes faster evolution of external sections and loss of homology in membrane proteins. Adaptation to new functions and niches causes faster evolution for outside-facing sections (top), potentially contributing to divergence beyond recognition. Other proteins may provide no advantage in the new environment, and could be lost entirely over time (centre). For simplicity, the species on the left is assumed to remain functionally identical to the common ancestor (bottom).

This principle is further supported by another experiment carried out by the authors, where they compared the rate of divergence and the degree of gene loss across the three domains of life and showed that indeed the order of the effect of these evolutionary forces is as predicted by the principle. However, the effect was still significant in multicellular eukaryotes.

This principle has great evolutionary, practical phylogenetics and medical implications. For instance, the positive selection pressure from recognition by the host results in a red-queen dynamics that drives faster evolution in MPs. Over half all known drug targets are MPs, which explains why many of the effective drugs in animal trials are unsuccessful in human trials. And last, but not least (at least to me!), if the MPs are less likely to be conserved across the tree of life, then homology searches and molecular clocks are more likely to be confounded. All of this points to the importance of the MPs in adaptability of organisms to new environment; especially microbial communities (not so gate-like after all!). All inferences, obviously, apply to transporters too, and I think, these inferences are even more exciting, in some ways, when the implications are revisited in the context of the importance of transporters in niche-adaptation. Who knows, I might write more extensively about my own views, and further investigations, regarding this matter, with a more specific emphasis on transporters.

Source: Sojo, Victor et al. “Membrane Proteins Are Dramatically Less Conserved Than Water-Soluble Proteins Across The Tree Of Life”. Molecular Biology and Evolution 33.11 (2016): 2874-2884.

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.

Using Single Molecule FRET to Understand Substrate Binding Domains

By Bryony Ackroyd

Twitter: @BryonyAckroyd

ABC transporters can be either import or export systems for cells. They consist of two transmembrane domains (TMDs) and two cytoplasmic nucleotide binding domains (NBDs). ABC importers also use substrate binding domains (SBDs) or substrate binding proteins (SBPs). SBPs are separate proteins present in the periplasm, however SBDs are fused to the TMDs. Some ABC transporters even have two or three SBDs fused together in tandem. Although this is a known phenomenon, very little is understood about the system and how ABC transporters are able to interact with multiple and structurally distinct SBDs. In the work carried out in this particular paper the group focusses on GlnPQ from L. lactis, a Gram-positive bacterium, that imports asapargine, glutamine and glutamate via two different SBDs.

Although there are crystal structures and NMR data available for SBDs, not much is known about the mechanism of ligand binding e.g. induced fit or conformational selection. Bearing this is mind Poolman et al., used a unique combination of techniques to probe the conformational dynamics of the SBDs, single-molecule Forster resonance energy transfer (smFRET) coupled with isothermal titration calorimetry (ITC). Using this strategy the group was able to provide mechanistic insight into the transport mechanisms of ABC importers, showing that the SBDs of GlnPQ bind ligands via an induced-fit mechanism.

The SBDs can be in one of four states, closed-ligand bound (CL), open (O), partially closed (PC) or closed (C). The induced-fit mechanism of binding triggers the CL state from the O state, however in the conformation-selection model the SBD can be in the PC or C states without a ligand bound. Ligand binding stabilises the PC state and therefore pushes the SBD to the CL conformation. These differing conformational states were examined via smFRET and the changes between states was observed via FRET efficiency. The experiment was designed so that the O conformation of the SBD gave a low FRET efficiency and the closed conformations gave higher FRET efficiency. Fluorophores were designed on the SBDs to be between 3-6 nm apart in both the closed and open states.

The SBD1 of GlnPQ binds asparagine with high affinity and glutamine with low affinity whereas SBD2 solely binds glutamine with a high affinity.

slide1
Single molecule dynamics of SBDs probed with smFRET. (a) Schematic showing immobilisation of histidine tagged SBDs to a PEG-biotin coated surface in a flow cell. The surface scan on the right is shown in flase colour, orange indicates double-labelled SBDs, green is SBDs with only donor fluorophore and red is SBDs with only acceptor fluorophores. (b-d) Representative fluorescence time traces, blue is donor signal, red acceptor signal, grey FRET signal and orange is the fit. These graphs show that the FRET efficiency of SBD1 and SBD2 increased as the concentration of substrate increased. This indicates that closing of SBD1 and SBD2 increased as substrate concentration increased, in keeping with the induced-fit model.

By measuring the fluorescence emitted from an SBD immobilised on a surface when varying concentrations of substrate were added, it was possible to determine the conformational state of the SBD and therefore whether the induced-fit or conformational-selection model was being employed by the SBD. Poolman et al., showed that in the absence of ligand the SBDs of GlnPQ were continuously in the O conformation and not in the PC or C conformations, therefore demonstrating the induced-fit model is used by the SBDs of GlnPQ.

This clever and unique technique was able to beautifully show the different conformations of the SBDs and conformational changes that occur within SBDs during ligand binding. Hopefully this technique will be employed more widely in the future to elucidate ligands, binding mechanisms and conformations of other SBDs and SBPs.

 

Source: Poolman et al., (2015). Conformational dynamics in substrate-binding domains influences transport in the ABC importer GlnPQ. Nature Structural and Molecular Biology 22, 57–64.