Final Report Summary - AGGREGAT3 (Protein dynamics and misfolding: the case study of a multidomain protein related to an amyloid disease)
The AggregAT3 project aimed to characterize the structural dynamic of the polyglutamine (polyQ) amyloid associated protein ataxin-3 (AT3). Aggregation of AT3 is the causative agent of Spinocerebellar Ataxia Type 3 (SCA3), a fatal neurodegenerative disorder which shares many common features with others amyloid related disorders. In the AT3 project we characterized for the first time in atomic details the dynamics associated with the structure of AT3 employing state of the art NMR techniques together with molecular dynamics simulation. In particular we demonstrated that the globular domain of AT3 presents a highly dynamic helical hairpin. The helical hairpin of AT3 is a key element for the physiological function of the protein as it dictates the solvent exposure of the ubiquitin binding region close to the catalytic site of the protein. Further, the ubiquitin binding site is also the major aggregation prone region of the globular domain. Therefore, movement of the hairpin is responsible for the access of a site that is key element for both the function and dysfunction of the protein. Our findings demonstrate that this helical hairpin is not constrained in an “open” conformation but due to large motions samples also “closed” conformations where the accessibility of the aggregation/binding region is largely diminished.
In AggregAT3 we also investigated the region spanning from the globular domain to the polyQ site of AT3. This region has a key role in AT3 aggregation as it greatly enhances and redirects the aggregation pathway of the globular domain leading to toxic aggregates able to trigger the symptoms of SCA3 in a murine model. However this region has not a well-defined structure and it has never been structurally characterized. We therefore by mean of a combined NMR, SAXS and MD approach structurally investigated this region. We showed that although being highly dynamic and disorder, this region has a high propensity to form alpha-helical structures. In particular, two well defined alpha-helices are present in two ubiquitin interaction motifs (UIMs) which were earlier identified by bioinformatics analysis. Also, this region showed to be more compact compared to an intrinsically disorder region of the same size and we were able to identify tertiary contact by the N-terminal part and the central part where the UIMs are located. The structural characterization of this part of the protein is particularly important as it paves the way to the study of the whole N-terminal part of AT3.
The AggregAT3 project traced the road to the ambitious future goal of the first structural and dynamical characterization of a multidomain protein involved in an amyloid disease. Starting from this project, we plan to investigate the effect of the disorder part on the dynamic of the globular folded part. In particular, it would be of great interest to characterize the effect on the motion of the helical hairpin or even other dynamic effects induced by the presence of this region. This could lead to depict the molecular reasons behind the effect of the disordered part on the AT3 aggregation, greatly enhancing our knowledge related to amyloid aggregation of multidomain proteins.