Flanking polyproline sequences inhibit β-sheet structure in polyglutamine segments by inducing PPII-like helix structure

Polyglutamine (poly(Q)) expansion is associated with protein aggregation into beta-sheet amyloid fibrils and neuronal cytotoxicity. In the mutant poly(Q) protein huntingtin, associated with Huntington's disease, both aggregation and cytotoxicity may be abrogated by a polyproline (poly(P)) domain flanking the C terminus of the poly(Q) region. To understand structural changes that may occur with the addition of the poly(P) sequence, we synthesized poly(Q) peptides with 3-15 glutamine residues and a corresponding set of poly(Q) peptides flanked on the C terminus by 11 proline residues (poly(Q-poly(P)), as occurs in the huntingtin sequence. The shorter soluble poly(Q) peptides (three or six glutamine residues) showed polyproline type II-like (PPII)-like helix conformation when examined by circular dichroism spectroscopy and were monomers as judged by size-exclusion chromatography (SEC), while the longer poly(Q) peptides (nine or 15 glutamine residues) showed a beta-sheet conformation by CID and defined oligomers by SEC. Soluble poly(Q)-poly(P) peptides showed PPII-like content but SEC showed poorly defined, overlapping oligomeric peaks, and as judged by CD these peptides retained significant PPII-like structure with increasing poly(Q) length. More importantly, addition of the poly(P) domain increased the threshold for fibril formation to approximate to 15 glutamine residues. X-ray diffraction, electron microscopy, and film CID showed that, while poly(Q) peptides with ! 6 glutamine residues formed beta-sheet-rich fibrils, only the longest poly(Q)-poly(P) peptide (15 glutamine residues) did so. From these and other observations, we propose that poly(Q) domains exist in a tug-of-war between two conformations, a PPII-like helix and a beta-sheet, while the poly(P) domain is conformationally constrained into a proline type 11 helix (PPII). Addition of poly(P) to the C terminus of a poly(Q) domain induces a PPII-like structure, which opposes the aggregation-prone beta-sheet. These structural observations may shed light on the threshold phenomenon of poly (Q) aggregation, and support the hypothesized evolution of protective poly(P) tracts adjacent to poly(Q) aggregation domains. (c) 2007 Elsevier Ltd. All rights reserved.