The formation of elongated, unbranched fibrillar protein aggregates, termed amyloid, has been linked to neurodegenerative diseases including Alzheimer’s Disease, Parkinson’s Disease and Amyotrophic Lateral Sclerosis (ALS). For decades, scientists have sought to determine the structures and polymorphic features of these aggregates to better understand their complex assembly and role in disease. Here, we show structures from three amyloid proteins, TAR DNA Binding Protein 43 (TDP-43), Superoxide Dismutase 1 (SOD1), and α-synuclein, offering insight into amyloid and protein aggregation in general.
First, we investigated TDP-43 as a model for the polymorphic capabilities of pathological amyloid aggregation. From x-ray diffraction, electron diffraction and cryoEM, we show that the segment, 247DLIIKGISVHI257, from the second RNA recognition motif forms an array of amyloid polymorphs. These associations include seven distinct interfaces displaying five different symmetry classes of steric zippers. The polymorphic nature of this segment illustrates at the molecular level how amyloid proteins can form diverse fibril structures.
In the second section, we investigated the role of the low complexity domain (LCD) of TDP-43 in reversible stress granule aggregation and irreversible pathogenic aggregation. We determined six atomic resolution segment structures from the LCD of TDP-43 that offer insight into regions of the protein responsible for aggregation. We illustrate how three of these structures, 300GNNQGSN306, 370GNNSYS375, and 396GFNGGFG402 form fibrils and exhibit characteristic steric zippers, indicative of pathogenic aggregation. We present an atomic structure for segment 312NFGAFS317, which reveals a fibril with a kinked interface that is reversible and may play a role in stress granule assembly. We present a model that combines previous literature with our new structural findings, to illustrate how stress granule assembly can lead to pathogenic aggregation.
Third, we structurally characterized peptide segments from SOD1 because pathological deposition of mutated SOD1 accounts for ~20% of the familial ALS (fALS) cases. We used a computational approach to discover four segments from the protein that form fibril-like aggregates and subsequently solved the structure of three of those segments: 101DSVISLS107, 147GVIGIAQ153 and 147GVTGIAQ153. We found, through the use of proline mutations, that two of these 101DSVISLS107 and 147GVIGIAQ153 are likely to trigger the aggregation of full-length SOD1, suggesting common molecular determinants of fALS and sALS.
In the fourth section, we investigated whether amyloid oligomers or fibrils were responsible for SOD1 toxicity in ALS. We determined the corkscrew-like structure of a cytotoxic segment of SOD1 in its oligomeric state. Through time course cytotoxicity assays, we demonstrated that the toxicity is likely a property of soluble oligomers and not large insoluble aggregates. Our work adds to evidence that the toxic oligomeric entities in protein aggregation diseases contain antiparallel, out-of-register β-sheet structures and identifies a target for structure-based therapeutics in ALS.
In the final section, we characterized the amyloidogenic core of α-synuclein, the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson’s disease. We utilized a new technique known as Micro-electron diffraction (MicroED) to solve the structure of crystals of an 11-residue segment we term NACore that are invisible by optical microscopy. The structure reveals protofibrils built of pairs of face-to-face β-sheets that exhibit a fiber diffraction pattern similar to full-length α-synuclein. We present a model of the toxic, full-length α-synuclein fibril, incorporating the NACore and an additional segment.
En masse, the structures in this dissertation offer broad insights into pathological and reversible protein aggregation. The structures provide a starting point for additional studies aimed at developing therapeutics for amyloid formation in neurodegenerative disease.