A star is born : development of NT* as a new biotechnological tool based on the mechanism of pH dependent dimerization of the spidroin N-terminal domain

Proteins are widely used in research and in the pharmaceutical industry but the production of recombinant protein can be tedious, costly and time-consuming due to unwanted aggregation. Strategies to circumvent aggregation, e.g. the use of solubility tags, must be evaluated experimentally and successful results are not always obtained. Therefore, there is a need to develop novel solubility tags for problematic proteins. In this thesis, we propose that spiders’ own solubility tag – the N-terminal domain (NT) of the spider silk proteins (spidroins) –– can be harnessed to produce very aggregation-prone proteins.

NT is a pH-sensitive relay and its conformational switch from monomer to dimer determine the assembly state of the spider silk proteins. However, the molecular mechanisms of NT dimerization are unclear. In this thesis we determined which residues regulate the dimerization of NT from major ampullate spidroin and investigated whether this mechanism is conserved between distantly related NTs. Our results showed that NT dimerization requires an initial electrostatic interaction mediated by aspartate 40 with lysine 65, and that upon lowering the pH, subsequent protonation of glutamates 79, 84 and 119 result in conformational changes and stabilization. Moreover, we provided evidence that charge attraction and multistep protonation is conserved between widely different NTs but is mediated by different sets of residues.

On the basis of these findings, we designed the soluble and thermally stable NT* by disrupting the charge attraction between aspartate 40 and lysine 65. NT* was more efficient to promote solubility of aggregation-prone proteins and peptides than commonly used solubility tags. We hypothesized further that NT* would be a suitable tag to control the solubility of amyloidogenic proteins and, thereby prevent precocious aggregation. Our results support that NT* prevents aggregation of amyloidogenic proteins, allowing e.g. structural studies and identification of new anti-amyloid strategies.

This thesis presents an entirely novel approach to produce aggregation-prone recombinant proteins using the biotechnological tool NT* which was conceived on the basis of how spiders store their spidroins at high concentration without precocious aggregation.

List of scientific papers

I. Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation. Kronqvist N., Otikovs M., Chmyrov V., Chen G., Andersson M., Nordling K., Landreh M., Sarr M., Jörnval H., Wennmalm S., Widengen J., Meng Q., Rising A., Otzen D., Knight S.D., Jaudzems K., Johansson J. Nature Communications. (2014); 5:3254.
https://doi.org/10.1038/ncomms4254

II. Conserved properties of spider silk protein N-terminal domain in spite of extensive sequence divergence. Sarr M., Kitoka K., Kaldmäe M., Walsh-White K.A., Landreh M., Jaudzems K., Rising A., Johansson J., Kronqvist N. [Manuscript]

III. Efficient protein production inspired by how spiders make silk. Kronqvist N., Sarr M., Lindqvist A., Nordling K., Otikovs M., Venturi L., Pioselli B., Purhonen P., Landreh M., Sjöberg L., Robinson C., Pelizzi N., Jörnvall H., Hebert H., Jaudzems., Curstedt T., Rising A., Johansson J. Nature Communications. (2017); 8:15504.
https://doi.org/10.1038/ncomms15504

IV. A spidroin-derived solubility tag enables controlled aggregation of a designed amyloid protein. Sarr M., Kronqvist N., Chen G., Aleksis, R., Purhonen P., Hebert H., Jaudzems K, Rising A., Johansson J. The FEBS Journal. (2018); 85(10):1873-1885.
https://doi.org/10.1111/febs.14451

V. High-yield production of amyloid-β peptide enabled by a customized spider silk domain. Abelein A., Chen G., Kitoka K., Aleksis R., Oleskovs F., Sarr M., Landreh M., Pahnke J., Nordling K., Kronqvist N., Jaudzems K., Rising A., Johansson J., Biverstål H. Scientific Reports. (2020); 10:235.
https://doi.org/10.1038/s41598-019-57143-x