Phosphorylation of α-synuclein fibrils at S129 changes DNAJB1 binding as probed by solid-state NMR
Presented by: Sayuri Pacheco
Abstract:
α-Synuclein (aSyn) is a soluble, dynamic protein in its native form, but in synucleinopathies such as Parkinson’s disease, it forms amyloid fibrils. These amyloid fibrils can be described by three main regions: the N-terminus with intermediate motions, the highly static fibril core, and the very dynamic C-terminus. Due to their solvent exposure and flexibility, the N- and C-termini—intrinsically disordered regions (IDRs) of aSyn fibrils—serve as targets for immunotherapies and as binding sites for many chaperone proteins.
DNAJB1, a chaperone protein, preferentially binds to the C-terminus of aSyn fibrils. It is part of a regulatory chaperone complex, formed with HSP70 and APG2, which collectively disaggregates misfolded and aggregated proteins. Although DNAJB1 does not disaggregate aSyn fibrils alone, it binds directly to the dynamic C-terminus, making this interaction an important subject of study. Previous research has focused primarily on the binding of DNAJB1 to wild-type (WT) aSyn fibrils. However, over 90% of aSyn fibrils in Lewy bodies are phosphorylated at Ser129. Therefore, we aimed to characterize the interaction between phosphorylated aSyn fibrils (pS129) and DNAJB1 in detail. Using biochemical assays and solid-state NMR (ssNMR), we demonstrated that DNAJB1 preferentially binds to pS129 fibrils. SsNMR further provided atomic-resolution insights into which residues are perturbed during this binding event. This research sheds light on the binding preferences of DNAJB1 and offers potential pathways for targeting protein aggregates in neurodegenerative diseases.
Additionally, our lab is characterizing the dynamics and residual structure of the IDRs of WT aSyn in both its monomeric and amyloid fibril states. Using ssNMR, EPR, and molecular dynamics (MD) simulations, we aim to understand how the IDRs change during fibril formation. Our ssNMR data demonstrate an increase in dynamics in the last 20 residues of the C-terminus of aSyn fibrils, allowing them to be detected with J-based NMR experiments. Continuous-wave (CW) EPR confirms that residues in the monomer are highly dynamic, whereas residues as early as residue 8 in the fibril are already semi-rigid and undetectable by ssNMR. These data are being used to validate our all-atom simulations, which will generate a conformational ensemble of structures that best represents a full-length aSyn fibril. This approach will enable us to pinpoint key differences between the IDRs in monomeric and fibrillar forms, shedding light on the distinct binding partners and properties of the two states.
