The polyglutamine amyloid nucleus in living cells is a monomer with competing dimensions of order

Abstract

A long-standing goal of the study of amyloids has been to characterize the physical nature of the rate-determining nucleating event. However, the transience and rarity of that event within the heterogeneous ensemble of states populated by amyloid-forming proteins make it inaccessible to classical biochemistry, structural biology, and computational approaches. Here, we address these limitations by measuring the dependence of amyloid formation on concentration and conformational templates in living cells, whose volumes are sufficiently small to resolve independent nucleation events. We characterized over one hundred rationally designed sequence variants of polyglutamine (polyQ), a polypeptide that precipitates Huntingtons and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. We deduce that amyloid formation by polyQ begins with a steric zipper embryo of approximately twelve interdigitated glutamine side chains within an individual polypeptide molecule. Formation of the embryo was limited to polypeptides longer than the pathogenic threshold, and involved neither phase separation nor oligomerization. We found that different amyloid propensities of polyQ sequence variants can be rationalized by steric zipper ordering orthogonally to the axis of polymerization, and validated this intuition using all-atom molecular dynamics simulations. The unique ability of the polyQ sequence to fold in this fashion not only allowed for polyQ amyloid to nucleate from low concentrations; it also stalled amyloid growth with a concomitant accumulation of partially-ordered oligomers. By illuminating the structural mechanism of polyQ amyloid formation in cells, our findings reveal a potential molecular etiology for polyQ diseases, and may provide a roadmap for the design of new therapies.

A nucleation barrier spring-loads the CBM signalosome for binary activation

“…To test if a high local concentration suffices for CARD9 CARD subunits to organize into BCL10 polymer nuclei, we substituted the coiled-coil region with an inert and well-characterized homomultimeric domain, μNS ( Schmitz et al, 2009 ). This domain forms stable condensates that drive its fusion partner to very high local concentration ( Figure 3A ) without affecting global expression levels ( Figure 3—figure supplement 2D ), and thereby eliminates intermolecular entropic contributions to nucleation barriers even at low expression ( Kandola et al, 2021 ). Expressing the CARD9 CARD -μNS-mCardinal fusion should trigger the high-AmFRET state of any yeast cell expressing either CARD9 CARD - or BCL10-mEos3.1 above their respective solubility thresholds.…”

Conformational entropy limits the transition from nucleation to elongation in amyloid aggregation

“…However, the sidechain interaction energy somewhat reduces the initial cost below 2Δ s and increases the attractive energy of subsequent molecules. The nucleation barrier can be further lowered along the two-sheet pathway if the molecules are long enough to form two β -strands (46).…”

“…However, the sidechain interaction energy somewhat reduces the initial cost below 2∆s and increases the attractive energy of subsequent molecules. The nucleation barrier can be further lowered along the two-sheet pathway if the molecules are long enough to form two β -strands (46). While our simulations only considered polyglutamine homopolymers, insight into the effect of amino acid sequence can be obtained from the barrier heights given by Eqs.…”

A nucleation barrier spring-loads the CBM signalosome for binary activation

“…To test if a high local concentration indeed suffices for CARD9 CARD subunits to organize into BCL10 polymer nuclei, we substituted the coiled coil region with an inert and well-characterized homomultimeric domain, μNS (Schmitz et al, 2009). This domain forms stable condensates that drive its fusion partner to very high local concentration (Fig 3A), and thereby eliminates intermolecular entropic contributions to nucleation barriers (Kandola et al, 2021). Expressing the CARD9 CARD -μNS fusion should force to high-AmFRET any cells expressing either CARD9 CARD - or BCL10-mEos3.1 above their respective concentration thresholds for polymerization.…”