Phosphorylation of α-synuclein fibrils at S129 changes DNAJB1 binding as probed by solid-state NMR

Amyloid fibrils composed of the protein α-synuclein (aSyn) are implicated in the pathogenesis of synucleinopathies. These pathological fibrils, characterized by their rigid amyloid cores, also feature flexible intrinsically disordered regions (IDRs) that interact with various cellular components. Due to their solvent exposure and flexibility, the N- and C-termini, IDRs of aSyn fibrils, have been used as targets for immunotherapies and serve as binding sites for many chaperones. Chaperones are known to play a vital role in preventing and reversing amyloid formation in neurodegenerative diseases, but how they recognize misfolded proteins is often poorly understood. DNAJB1 is a co-chaperone that is part of a larger complex formed with Hsp70 and Apg2, which collectively disaggregate amyloid fibrils such as those formed by aSyn, tau, or huntingtin. Although the entire chaperone and co-chaperone complex are required for aggregate disassembly, DNAJB1 directly recognizes these amyloid fibrils. More specifically, DNAJB1 preferentially binds to the C-terminus of wild-type aSyn. However, over 90% of aSyn fibrils in Lewy bodies are phosphorylated at S129. Here, we determine the effect of S129 phosphorylation on DNAJB1-aSyn fibril binding using biochemical assays and solid-state NMR. We show that DNAJB1 preferentially binds aSyn pS129 fibrils and that the aSyn binding site of pS129 and wild-type fibrils is different. These results suggest that pS129 might influence chaperone-mediated degradation efficiency.