A “spindle and thread”-mechanism unblocks translation of N-terminally disordered proteins

Written by: Mathys Couperus and Anna Knuistingh Neven

Background
The tumour suppressor protein p53 is a key regulator that plays an essential role in cell proliferation, apoptosis and the repair of damaged DNA. The p53 pathway is crucial in maintaining a potent barrier against cancer development. Inactivation or dysfunction of the protein is disastrous, and mutations that inactivate p53 can be found in most human cancers. Consequently, stabilising p53 is an attractive therapeutic strategy in combating cancer.

The protein p53 is not particularly known for its stable three-dimensional structure.  On the contrary, the short half-life of p53 leads to low expression levels in human cells, providing marginal evolutionary pressure to adopt a stable conformation. P53 has a high tendency to form aggregates with itself and other proteins (Silva et al., 2014). Several mutated p53 variants show prion-like behaviour and have a higher propensity to aggregate, leading to tissue invasion, rapid proliferation and metastasis (Muller et al., 2013).

Human proteins with intrinsically disordered domains generally display significantly shorter half-lives than proteins without these domains (van der Lee et al., 2014). Fusion to a non-dimerizing mutant of the highly conserved N-terminus of a spider silk protein (NT*) enables efficient production of aggregation-prone peptides (Abelein et al., 2020; Kronqvist et al., 2017; Sarr et al., 2018). The authors of this preprint hypothesise that generating a p53 mutant utilising NT* could lead to new structural and functional insights into protein chemistry.

 

Results
Kaldmäe et al. investigate the effect of NT* on p53 translation by using three constructs with a C-terminal GFP tag, that compass either a NT* domain, full length p53 or NT* fused to p53. In vitro transcription and translation of NT*-GFP shows a massive increase in GFP fluorescence (Figure 1C from the preprint). Comparing translation levels of p53 with either a N-terminal or C-terminal NT* using SDS-PAGE reveals the importance of the location of the NT* tag. Only the N-terminally tagged protein can be detected, suggesting that an N-terminal NT* increases translation of p53. This raises the question whether the difference in GFP fluorescence between p53-GFP and NT*-p53-GFP could be due to differences in ribosome activity. Polysome profiling shows that the constructs with the fused NT* has three times more translating ribosomes compared to p53-GFP RNA, which indicates that translation of p53 is either slowed down or stalled in the absence of NT* (Figure 1E).

 

Next, the authors examine the effect of NT* on the folding of p53. Glutaraldehyde crosslinking and Western Blot analysis confirm that the NT* domain does not alter the oligomeric state of the p53 moiety. All-atom molecular dynamics simulations on the first 90 residues of p53, comprising the TAD and PRD domains, show a shift in the RMSF (root mean square fluctuation) in the presence of NT* (Fig. 2D). These fluctuations are caused by the fact that the p53 polypeptide wraps around the NT* domain (Fig. 2E). Thus, the presence of NT* significantly reduces disorder from the intrinsically disordered N-terminus of p53.

Folding of the p53 TADs and PRD onto NT* is likely driven by hydrophobic collapse. The hydrophobic residues of TAD1 interact with a mainly hydrophobic patch on the surface of NT*, which is part of the dimerization interface in the wildtype NT-domain (Fig. 2E). Ion mobility mass spectrometry confirms the interaction by measuring the collision cross sections and relative abundances. These findings together indicate that NT* remains folded in the p53 fusion protein and induces a compact conformation of the chimeric construct, in which the p53 N-terminal transactivation region wraps around NT*.

 

The findings thus far raise the question whether the approach of introducing a more compatible N-terminus for a partially disordered protein work on other proto-oncogenes as well. The serine/threonine-protein kinase B-Raf contains a disordered N-terminal region followed by a folded domain, like p53. When fused with NT* in the same way as done for NT*-p53, the NT* tagged protein is readily expressed in high amounts, while the untagged protein cannot be detected using SDS-PAGE analysis. The GTPase K-Ras contains a stable folded N-terminus, in contrast to p53 and B-Raf. As expected, the production of K-Ras is not affected by N-terminal addition of NT*, showing high expression levels independently of being fused to an NT* domain.

 

Main message and future implications

The specific properties of NT* increase the poor translation efficiency of disordered N-terminal proteins like p53 and B-Raf. The chimeric p53 protein shows a 3-fold increase in expression and adopts a more compact conformation. This discovery may benefit therapeutic strategies using synthetic mRNAs, because the NT*-mediated translation efficiency would reduce the number of mRNAs that must be administered to get the desired effect.

 

What we like about this preprint

We find it remarkable that the authors used the N-terminus from a spider silk protein to make p53 more stable and increasing its translation. The proteins in spider silk are one of the most stable and strong natural compounds. It is an interesting concept to combine these stable domains with an unstable human protein such as p53. By making p53 more stable, it will become less aggregation prone. Aggregation of proteins is a hallmark of many diseases. The findings are exciting in a broader sense because they shed more light on how inducing co-translational folding can overcome poor translation efficiency of partially disordered proteins.

 

Questions to authors

1. It is interesting that when compared to the WT p53, relatively more NT*-p53 is found in the cytoplasm. The p53 protein shows different regulatory properties based on its location, be it nucleus or cytoplasm. If transport of the variant protein is less controllable than its WT counterpart, would that not affect the biological activity of the engineered protein?
2. It is fascinating that the addition of an engineered stable N-terminus found in spider silk proteins increases the translation of p53. Given that in healthy cells p53 is degraded quickly, would the increased stability not be detrimental for the cell?

 

Reference list

• Abelein, A., Chen, G., Kitoka, K., Aleksis, R., Oleskovs, F., Sarr, M., Landreh, M., Pahnke, J., Nordling, K., Kronqvist, N., et al. (2020). High-yield Production of Amyloid-β Peptide Enabled by a Customized Spider Silk Domain. Sci. Rep. 10, 235.
• Kronqvist, N., Otikovs, M., Chmyrov, V., Chen, G., Andersson, M., Nordling, K., Landreh, M., Sarr, M., Jörnvall, H., Wennmalm, S., et al. (2014). Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation. Nat. 5, 3254.
• Muller, P., Vousden, K. (2013). p53 mutations in cancer. Nature Cell Biology, 15, 2–8.
• van der Lee, R., Lang, B., Kruse, K., Gsponer, J., de Groot, N.S., Huynen, M.A., Matouschek, A., Fuxreiter, M., and Babu, M.M. (2014). Intrinsically disordered segments affect protein half-life in the cell and during evolution. Cell Rep. 8, 1832–1844.
• Sarr, M., Kronqvist, N., Chen, G., Aleksis, R., Purhonen, P., Hebert, H., Jaudzems, K., Rising, A., and Johansson, J. (2018). A spidroin-derived solubility tag enables controlled aggregation of a designed amyloid protein. FEBS J. 285, 1873–1885.
• Silva, J.L., Gallo, C.V.D.M., Costa, D.C.F., and Rangel, L.P. (2014). Prion-like aggregation of mutant p53 in cancer. Trends Bioch Sci. 39, 260–267.

Tags: intrinsic disorder, p53, protein engineering, spidroins

doi: Pending

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