Structural characterization of the functional amyloid protein Orb2A, and evaluation of structure-based inhibitors of amyloid assembly
Amyloids are stable protein assemblies characterized by their β-sheet rich secondary structure and unbranched, fibrillar morphology. Whereas formation of amyloid is traditionally associated with neurodegenerative diseases such as Alzheimer’s disease (AD), the amyloid fold has also been adapted for beneficial biological functions. These so-called functional amyloids exhibit similar structural characteristics to pathogenic amyloids, but are not toxic to their hosts. The study of functional amyloid structure and mechanisms may provide insight into amyloid disease pathogenesis, and aid in the development of therapeutics. In chapter one, I present the atomic-resolution structure of the amyloid-driving N-terminal segment of the functional amyloid protein Orb2A. Using micro-electron diffraction (micro-ED), I determined the structure of this nine-residue segment, which I term M9I, and found that it forms a classical amyloid steric zipper structure, with phenylalanine side chains playing a critical role in the formation of the self-complementary dry interface. Using electron microscopy, x-ray diffraction, and Thioflavin-T binding assays I show that the M9I segment is sufficient to form amyloid-like fibrils, and replacement of phenylalanine residues with tyrosine reduces fibril formation of the Orb2A prion-like domain (PLD). I also propose a structural model for full-length Orb2A that incorporates both this M9I steric zipper structure, and a previously published cryo-EM structure of the downstream glutamine/histidine (Q/H)-rich region from the related Orb2B isoform. In chapters two and three, I evaluate amyloid aggregation inhibitors that were rationally designed using steric zipper structures of segments derived from full-length proteins. Chapter two describes the amyloidogenic and phase-separating behavior of the nucleocapsid (NCAP) protein of SARS-CoV-2, and the structure-based design of peptide inhibitors against steric zipper-forming segments from its central low-complexity domain (LCD). My contribution to this work included high-throughput screening of this inhibitor panel in a cell culture model of SARS-CoV-2 infection, and subsequent evaluation of inhibitor hits. I identified an inhibitor termed G12 that robustly reduced SARS-CoV-2 infection in a dose-dependent manner, and disrupted phase-separation of NCAP in vitro; other weaker inhibitors of SARS-CoV-2 infection had no effect on NCAP phase separation, thereby correlating disruption of NCAP condensation with reduced infection in cultured cells. This work demonstrates that amyloid fibrils formed by NCAP can be targeted for antiviral drug design, and the G12 peptide inhibitor is a prototype molecule that may be further optimized for the treatment of severe COVID-19 disease. Chapter three describes the structure of the AD-associated protein amyloid-β (residues 16-26 D23N), and the design of peptide inhibitors that reduce cytotoxicity and aggregation of full-length Aβ(1-42) and cross-seeding of tau by Aβ. My contribution to this work was in evaluation of inhibitor panels for reduction of Aβ-induced cytotoxicity in N2a cells. I identified one inhibitor termed D1 that reduced cytotoxicity when co-incubated with Aβ(1-42) prior to exposure to cells; a second generation of inhibitors was designed based on D1, and I identified two (D1b and D1d) that reduced cytotoxicity both when co-incubated with Aβ, and when added to pre-formed Aβ fibers directly before exposure to N2a cells. These inhibitors may be considered as lead molecules that can be further optimized for treatment of AD.