Mutational analysis of the structural organization of polyglutamine aggregates

Abstract

The formation of amyloid-like aggregates by expanded polyglutamine (polyGln) sequences is suspected to play a critical role in the neuropathology of Huntington's disease and other expanded CAG-repeat diseases. To probe the folding of the polyGln sequence in the aggregate, we replaced Gln–Gln pairs at different sequence intervals with Pro–Gly pairs, elements that are compatible with β-turn formation and incompatible with β-extended chain. We find that PGQ 9 and PGQ 10 , peptides consisting of four Q 9 or Q 10 elements interspersed with PG elements, undergo spontaneous aggregation as efficiently as a Q 45 sequence, whereas the corresponding PGQ 7 and PGQ 8 peptides aggregate much less readily. Furthermore, a P D GQ 9 sequence containing d -prolines aggregates more efficiently than the peptide with l -prolines, consistent with β-turn formation in aggregate structure. Introduction of one additional Pro residue in the center of a Q 9 element within PGQ 9 completely blocks the peptide's ability to aggregate. This strongly suggests that the Q 9 elements are required to be in extended chain for efficient aggregation to occur. We determined the critical nucleus for aggregation nucleation of the PGQ 9 peptide to be one, a result identical to that for unbroken polyGln sequences. The PGQ N peptide aggregates are structurally quite similar to Q 45 aggregates, as judged by heterologous seeding aggregation kinetics, recognition by an anti-polyGln aggregate antibody, and electron microscopy. The results suggest that polyGln aggregate structure consists of alternating elements of extended chain and turn. In the future it should be possible to conduct detailed and interpretable mutational studies in the PGQ 9 background.