β-Hairpin-Mediated Nucleation of Polyglutamine Amyloid Formation

Abstract

The conformational preferences of polyglutamine (polyQ) sequences are of major interest because of their central importance in the expanded CAG repeat diseases that include Huntington’s disease (HD). Here we explore the response of various biophysical parameters to the introduction of β-hairpin motifs within polyQ sequences. These motifs (trpzip, disulfide, D-Pro-Gly, Coulombic attraction, L-Pro-Gly) enhance formation rates and stabilities of amyloid fibrils with degrees of effectiveness well-correlated with their known abilities to enhance β-hairpin formation in other peptides. These changes led to decreases in the critical nucleus for amyloid formation from a value of n* = 4 for a simple, unbroken Q23 sequence to approximate unitary n* values for similar length polyQs containing β-hairpin motifs. At the same time, the morphologies, secondary structures, and bioactivities of the resulting fibrils were essentially unchanged from simple polyQ aggregates. In particular, the signature pattern of SSNMR 13C Gln resonances that appears to be unique to polyQ amyloid is replicated exactly in fibrils from a β-hairpin polyQ. Importantly, while β-hairpin motifs do produce enhancements in the equilibrium constant for nucleation in aggregation reactions, these Kn* values remain quite low (~ 10−10) and there is no evidence for significant embellishment of β-structure within the monomer ensemble. The results indicate an important role for β-turns in the nucleation mechanism and structure of polyQ amyloid and have implications for the nature of the toxic species in expanded CAG repeat diseases.

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

“…S1 shows that these NMR signals are indistinguishable from those of "simple" polyQ fibrils without htt flanking domains (11,(15)(16)(17). Previous studies have noted that these chemical shifts are indicative of β-sheet rather than α-helical or random coil structure.…”

“…Although it is generally accepted that the mature fibrils feature antiparallel β-sheets, there continue to be conflicting models not only for their fibrillar structure, but also for the specifics of the multistage pathway by which they are formed. For instance, intramolecular polyQ-based β-hairpins are both proposed to be either nuclei that initiate the rapid formation of β-hairpinbased fibrils (11) or semistable monomeric or oligomeric species that at least transiently resist progression to fibrils (12). Concrete structural data on the fibril's internal structure are essential prerequisites for a truly molecular understanding of the way in which mutant htt exon1 and other polyQ disease proteins misfold and aggregate.…”

“…We previously used MAS ssNMR to elucidate the domain structure of aggregated htt N-terminal fragments, in which we identified the rigid amyloid core (15,16). This polyQ-based amyloid core was found to have an unusual spectral signature that is also shared by other polyQ aggregates (11,15,17). However, until now, no ssNMR-based structural constraints on the htt exon1 amyloid core were available.…”

Aggregation landscapes of Huntingtin exon 1 protein fragments and the critical repeat length for the onset of Huntington’s disease

“…Structural characterization of the aggregates (13,14,(18)(19)(20) has shown that, even when there are flanking sequences, polyQ remains the fiber core and adopts a β-hairpin conformation. In this paper, we use energy landscape analysis to provide a detailed molecular picture of the aggregation process of the peptide encoded by HTT exon 1, focusing on how the flanking sequences influence aggregation.…”

“…The aggregation of the diseasecausing peptide is, however, further complicated by the presence of flanking amino acid sequences in fragments encoded by HTT exon 1. Experiments indicate that the addition of NT17 at the N terminus of polyQ enormously accelerates the aggregation, probably by encouraging the formation of prefibrillar oligomers (10)(11)(12)(13)(14)(15)(16), whereas the addition of the proline-rich region at the C terminus decreases the rate of aggregation apparently without changing fundamentally the mechanism (10,16,17). Structural characterization of the aggregates (13,14,(18)(19)(20) has shown that, even when there are flanking sequences, polyQ remains the fiber core and adopts a β-hairpin conformation. In this paper, we use energy landscape analysis to provide a detailed molecular picture of the aggregation process of the peptide encoded by HTT exon 1, focusing on how the flanking sequences influence aggregation.…”