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

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.