495.1 - Structure of a pathologic amyloid nucleus determined by rational genetic deconstruction of an intracellular nucleation barrier

Randal Halfmann (Stowers Institute for Medical Research, Stowers Institute for Medical Research), Tej Kandola (Stowers Institute for Medical Research, Stowers Institute for Medical Research), Shriram Venkatesan (Stowers Institute for Medical Research), Jaihui Zhang (North Carolina State University), Brooklyn Lerbakken (Stowers Institute for Medical Research), Jillian Blanck (Stowers Institute for Medical Research), Jianzheng Wu (Stowers Institute for Medical Research), Jay Unruh (Stowers Institute for Medical Research), Paula Berry (Stowers Institute for Medical Research), Jeffrey Lange (Stowers Institute for Medical Research), Alex Von Schulze (Stowers Institute for Medical Research), Andrew Box (Stowers Institute for Medical Research), Malcolm Cook (Stowers Institute for Medical Research), Celeste Sagui (North Carolina State University)

A long-standing goal of the study of amyloids has been to characterize the structural basis of the rate-determining nucleating event. However, the ephemeral nature of that event has made it inaccessible to classical biochemistry, structural biology, and computational approaches. Here, we addressed that limitation by using Distributed Amphifluoric FRET to measure the dependence of amyloid formation on concentration and conformational templates in living cells, whose volumes are sufficiently small to resolve the outcomes of independent nucleation events. We characterized numerous rationally designed sequence variants of polyglutamine (polyQ), a polypeptide that precipitates Huntington’s and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. This effort uncovered a pattern of approximately twelve Qs, only for polypeptides exceeding the clinical length threshold, that allow for amyloid nucleation to occur spontaneously within single polypeptides. Nucleation was inhibited by intermolecular phase separation. Using atomistic molecular dynamics simulations, we found that the pattern encodes a minimal steric zipper of interdigitated side chains. Lateral growth of the steric zipper competed with axial growth to produce “pre-amyloid” 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.