Polyglutamine Amyloid Core Boundaries and Flanking Domain Dynamics in Huntingtin Fragment Fibrils Determined by Solid-State Nuclear Magnetic Resonance

Abstract

In Huntington’s disease, expansion of a polyglutamine (polyQ) domain in the huntingtin (htt) protein leads to misfolding and aggregation. There is much interest in the molecular features that distinguish monomeric, oligomeric, and fibrillar species that populate the aggregation pathway and likely differ in cytotoxicity. The mechanism and rate of aggregation are greatly affected by the domains flanking the polyQ segment within exon 1 of htt. A “protective” C-terminal proline-rich flanking domain inhibits aggregation by inducing polyproline II structure (PPII) within an extended portion of polyQ. The N-terminal flanking segment (httNT) adopts an α-helical structure as it drives aggregation, helps stabilize oligomers and fibrils, and is seemingly integral to their supramolecular assembly. Via solid-state nuclear magnetic resonance (ssNMR), we probe how, in the mature fibrils, the htt flanking domains impact the polyQ domain and in particular the localization of the β-structured amyloid core. Using residue-specific and uniformly labeled samples, we find that the amyloid core occupies most of the polyQ domain but ends just prior to the prolines. We probe the structural and dynamical features of the remarkably abrupt β-sheet to PPII transition and discuss the potential connections to certain htt-binding proteins. We also examine the httNT α-helix outside the polyQ amyloid core. Despite its presumed structural and demonstrated stabilizing roles in the fibrils, quantitative ssNMR measurements of residue-specific dynamics show that it undergoes distinct solvent-coupled motion. This dynamical feature seems reminiscent of molten-globule-like α-helix-rich features attributed to the nonfibrillar oligomeric species of various amyloidogenic proteins.

MAS 1 H NMR Probes Freezing Point Depression of Water and Liquid-Gel Phase Transitions in Liposomes

“…3 B). A similar reduction in the water freezing temperature is commonly observed in MAS ssNMR studies of biological samples (42)(43)(44)(45). For instance, MAS NMR studies on tightly packed protein crystals showed that the bulk water did not completely freeze until below À15 C, whereas crystal waters remained unfrozen until À25 C (42).…”

“…As a consequence, ice formation happens first in the extravesicular bulk water, well before the intravesicular or interlamellar water pools freeze. This is analogous to the bulk and protein-associated water pools in crystalline and fibrillar protein samples (42)(43)(44)(45). For lipid vesicles or cellular preparations, the formation of extravesicular (or extracellular) ice leads to both a dehydration and a disruption of the lipid bilayer.…”

“…More importantly, protocols for preparing MAS ssNMR samples typically maximize signalto-noise by densely packing the (nonaqueous) material of interest: lipids, proteins, or other (bio)materials. In our studies (29)(30)(31)45,49), this is routinely accomplished by pelleting membrane or fibril samples in an ultracentrifuge. In the resulting sample, the amount of ''bulk'' water is purposely minimized, while (even transient) sample dehydration is reliably avoided.…”

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

“…Mutant htt exon1 with an expanded 44-residue polyQ domain was expressed as a maltose-binding protein (MBP) fusion construct (Fig. 1A) (5,16). Cleavage with factor Xa releases exon1, which first forms oligomeric aggregates (Fig.…”

“…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).…”