Ataxin-3 (At3) is the causative protein of the neurodegenerative disease spinocerebellar ataxia type-3, also known as Machado-Joseph disease. At3 is a multi-domain polyglutamine protein composed of a globular N-terminal catalytic domain, the Josephin domain, and a C-terminal region containing two UIMs, the polyglutamine tract, and a hydrophobic C-terminus. Expansion of the polyglutamine tract beyond 45-50 repeats results in neurodegenerative disease characterised by At3 misfolding and the accumulation of aggregates in intra-neuronal inclusions. In order to develop therapies it is important to understand the precise mechanisms that result in these misfolding events. At3 has a well characterised aggregation mechanism, but the link between polyglutamine expansion and pathogenesis remains unclear.

Previous work has shown that the expansion of the polyglutamine tract does not alter the stability of At3. Here we use alanine and peptide scanning to map regions within the Josephin domain that instigate the earliest events of the aggregation pathway. In parallel, we use hydrogen-deuterium exchange (HDX) coupled with reverse-phase HPLC and mass spectrometry to study the dynamics of At3 in its native, monomeric form. Remarkably, as polyglutamine length is increased, there is a corresponding increase in dynamics within the Josephin domain. Point mutations within the Josephin domain both slow down the aggregation rate and show decreased dynamics by HDX analysis. As such, we show for the first time that polyglutamine expansion changes the dynamics of the Josephin domain of At3 in a manner dependent of the presence and length of the polyglutamine tract. Therefore, by stabilising these dynamics, the propensity to aggregate can be reduced without altering the global thermodynamic stability of the protein.