Intrinsically disordered domains of α-synuclein: effect of fibril formation and fibril polymorphs - Many high-resolution structures of the cross-β core of amyloid fibrils have been solved using solid-state NMR and cryo-electron microscopy in recent years. However, most fibrils important in neurodegenerative diseases, as for example α-synuclein (aSyn) fibrils found in in Lewy body dementia (LBD) and other synucleinopathies, have sizable intrinsically disordered regions (IDRs) surrounding their ordered cross-β fibril core. These IDRs are part of the fibril surface, where they can interact with fibril-specific binders and be important for fibril toxicity. Therefore, determining the residual structure and dynamics of these IDRs, how they interact with other cellular components, and how they relate to fibril toxicity is the next logical step in the LBD and Alzheimer’s disease related dementia (ADRD) field. The goal of this proposal is to determine the conformational ensemble and dynamics of the IDRs from aSyn fibrils important in LBD, ADRD and other synucleinopathies. The N and especially the C-terminus of aSyn are intrinsically disordered in the fibril. These IDRs are binding sites for fibril-specific interactors such as the co-chaperone DNAJB1. Our central hypothesis is that a specific fibril core structure (polymorph) will determine the residual structure and dynamics of these IDRs and consequently the interaction with fibrils-specific binders. The rationale of this research is that only complete molecular models of amyloid fibrils that include their IDRs will allow us to determine polymorph-specific binding partners, which can explain the difference between LBD and other synucleinopathies. These complete molecular models will not only point to natural interactors, but also to disease-specific biomarkers and therapeutics for LBD as well as ADRD and other synucleinopathies. We will use a combination of solid-state NMR, EPR, and molecular dynamics simulations to test our hypothesis using three specific aims. Aim 1 is to determine the change in residual structure and dynamics of IDRs upon fibril formation. Based on the known core structure and our conformational ensemble of the IDRs, we will create a model of the entire fibril. Aim 2 is to determine the effect of fibril polymorphs on residual structure and dynamics of IDRs. We will show to what degree a specific cross-β core determines the conformational ensemble of its adjacent IDRs and determine the cytotoxicity of different aSyn polymorphs and chimeras. Aim 3 is to determine the effect of the fibril core on aSyn-DNAJB1 interaction. Here, our hypothesis is that fibril formation increases the accessibility of the DNAJB1 binding site and that some fibril cores found in LBD and other synucleinopathies do this better than others. These aims will (i) determine the changes in the IDRs outside the fibril core upon fibril formation and result in a whole fibril model. We will (ii) learn how specific cross-β core structures found in LBD and other synucleinopathies change the conformational ensemble and dynamics of the IDRs, and (iii) we will understand how these changes influence the interaction of specific binders, in our case DNAJB1. Together these advances will facilitate the development of new approaches to diagnose and treat LBD and ADRD.
