Unconjugated
A 21-nucleotide duplication in one allele of SNCA was identified in a previously described disease with abundant a-synuclein inclusions that we now call juvenile-onset synucleinopathy (JOS). This mutation translates into the insertion of MAAAEKT after residue 22 of a-synuclein, resulting in a protein of 147 amino acids. Both wild-type and mutant proteins were present in sarkosyl-insoluble material that was extracted from frontal cortex of the individual with JOS and examined by electron cryo-microscopy. The structures of JOS filaments, comprising either a single protofilament, or a pair of protofilaments, revealed a new a-synuclein fold that differs from the folds of Lewy body diseases and multiple system atrophy (MSA). The JOS fold consists of a compact core, the sequence of which (residues 36-100 of wild-type a-synuclein) is unaffected by the mutation, and two disconnected density islands (A and B) of mixed sequences. There is a non-proteinaceous cofactor bound between the core and island A. The JOS fold resembles the common substructure of MSA Type I and Type II dimeric filaments, with its core segment approximating the C-terminal body of MSA protofilaments B and its islands mimicking the N-terminal arm of MSA protofilaments A. The partial similarity of JOS and MSA folds extends to the locations of their cofactor-binding sites. In vitro assembly of recombinant wild-type a-synuclein, its insertion mutant and their mixture yielded structures that were distinct from those of JOS filaments. Our findings provide insight into a possible mechanism of JOS fibrillation in which mutant a-synuclein of 147 amino acids forms a nucleus with the JOS fold, around which wild-type and mutant proteins assemble during elongation.
