Structural basis for the interactions of amyloids with their cofactors
OA Version
Citation
Abstract
Amyloid diseases involve the pathological deposition of normally soluble proteins or peptides as insoluble fibrils in various organs, such as the brain, heart, liver, or lungs, causing organ damage. These deposits not only contain fibrillar proteins, but also other components such as non-fibrillar auxiliary proteins, glycosaminoglycans, lipids, nucleic acids, or metal ions. Although these non-fibrillar components are constitutively found in amyloid deposits and have been found to affect amyloid fibril growth, the structural basis for their interaction is unclear. In this dissertation, a wide variety of biophysical and computational techniques are used to investigate the structural basis for the interaction of amyloid fibrils with their cofactors. Computationally, I used flexible protein-heparin docking to investigate the structural basis for the interaction of various amyloid fibrils with glycosaminoglycans, which revealed that negatively charged heparin binds vertically to in-register arrays of positively charged residues in amyloid fibrils. This binding mode suggests that glycosaminoglycans may increase amyloid formation by satisfying the uncompensated positive charge of these basic residues, which is energetically unfavorable. Protein-protein docking of apolipoprotein E with a repertoire of patient-derived amyloid-β fibril polymorphs coupled with molecular dynamics of selected apolipoprotein E-amyloid-β fibril complexes revealed a hydrophobic binding mode facilitated by sequential inter- and intradomain rearrangement of apolipoprotein E, which is reminiscent of apolipoprotein E lipid binding. This binding mode was consistent for docking of other apolipoproteins, such as apoC-III and the consensus sequence peptide, which suggests there is a common amyloid-apolipoprotein hydrophobic binding mode. Experimentally, I used circular dichroism spectroscopy, hydrogen-deuterium exchange mass spectrometry, and thioflavin T fluorescence kinetics to investigate the effect of glycosaminoglycans and phospholipids on protein secondary structure, local conformation, and amyloid formation kinetics, using serum amyloid A as a model amyloid protein. I completed these studies using heparin as a model glycosaminoglycan and phosphatidylethanolamine and phosphatidylcholine as model phospholipids. The results of this dissertation provide a structural basis for how various amyloid cofactors interact with amyloid fibrils. The results also help to explain how amyloid polymorphism may contribute to confounding experimental results regarding the effect of amyloid cofactors on amyloid formation.
Description
2024