Ilag, L. I. | Search bioRxiv

bioRxiv2021

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

Protein disorder is a major hurdle for structural biology. A prominent example is the tumour suppressor p53, whose low expression levels and poor conformational stability due to a high degree of disorder pose major challenges to the development of cancer therapeutics. Here, we address these issues by fusing p53 to an engineered spider silk domain termed NT*. The chimeric protein displays highly efficient translation in vitro and in E. coli and is fully active in human cancer cells. The transmission electron microscopy structure and native mass spectrometry reveal that the full-length p53 fusion protein adopts a compact conformation. Molecular dynamics simulations show that the disordered transactivation domain of p53 is wound around the NT* domain via a series of folding events, resulting in a globular structure. We find that expression of B-Raf, another partially disordered cancer target, is similarly enhanced by fusion to NT*. In summary, we demonstrate how inducing co-translational folding via a molecular "spindle and thread" mechanism can overcome poor translation efficiency of partially disordered proteins.

biochemistry↗

bioRxiv2020

Amyloid-β oligomers are captured by the DNAJB6 chaperone: Direct detection of interactions that can prevent primary nucleation

A human molecular chaperone protein, DNAJB6, is an efficient inhibitor of amyloid aggregation owing to a unique motif with conserved S/T-residues with high capacity for hydrogen bonding. Global analysis of kinetics data previously showed that especially the primary nucleation rate is inhibited. It was concluded that DNAJB6 achieves this remarkably effective and sub-stoichiometric inhibition by interacting not with the monomeric unfolded conformations of the amyloid-{beta} (A{beta}) peptide but with aggregated species. The pre-nucleation oligomeric aggregates are transient and difficult to study experimentally. Here we employed an approach to directly detect oligomeric forms of A{beta} formed in solution by subsequent analysis with native mass spectrometry (native MS). Results show that the signals from the various forms of A{beta} (1-40) oligomers were reduced considerably in the presence of DNAJB6, but not with a mutational variant of DNAJB6 in which the S/T-residues were substituted. With focus on DNAJB6 we could also detect signals that appear to represent DNAJB6 dimers and trimers to which varying amounts of A{beta} is bound. These data provide direct experimental evidence that it is the oligomeric forms of A{beta} that are captured by DNAJB6 in a manner which is dependent on the S/T residues. Strong binding of A{beta} oligomers to DNAJB6 should indeed inhibit the formation of amyloid nuclei, in agreement with the previously observed decrease in primary nucleation rate.

biochemistry↗