Structurally targeted mutagenesis identifies key residues supporting α-synuclein misfolding in multiple system atrophy.

BACKGROUND

Multiple system atrophy (MSA) and Parkinson's disease (PD) are caused by misfolded α-synuclein spreading throughout the central nervous system. While familial PD is linked to several α-synuclein mutations, no mutations are associated with MSA. We previously showed that the familial PD mutation E46K inhibits replication of MSA prions both and , providing key evidence to support the hypothesis that α-synuclein adopts unique strains in patients.

OBJECTIVE

Here we sought to further interrogate α-synuclein misfolding to identify the structural determinants that contribute to MSA strain biology.

METHODS

We engineered a panel of cell lines harbouring both PD-linked and novel mutations designed to identify key residues that facilitate α-synuclein misfolding in MSA. We also used Maestro analyses to predict the effect of each mutation on α-synuclein misfolding into one of the reported MSA cryo-electron microscopy conformations.

RESULTS

In many cases, our modelling accurately identified mutations that facilitated or inhibited MSA replication. However, Maestro was occasionally unable to predict the effect of a mutation, demonstrating the challenge of using computational tools to investigate intrinsically disordered proteins. Finally, we used our cellular models to determine the mechanism underlying the E46K-driven inhibition of MSA replication, finding that the E46/K80 salt bridge is necessary to support α-synuclein misfolding.

CONCLUSIONS

Our studies used a structure-based approach to investigate α-synuclein misfolding, resulting in the creation of a powerful panel of cell lines that can be used to interrogate MSA strain biology.