Deciphering the Molecular Recognition of α-Synuclein by the Hsp40-Hsp70 Chaperone Machinery

The molecular chaperone network composed of Hsp40 and Hsp70 plays a central role in maintaining proteostasis by preventing protein misfolding, promoting refolding, and facilitating the clearance of protein aggregates. In Parkinson’s disease, the intrinsically disordered protein α-Synuclein undergoes aberrant self-association into toxic oligomers and amyloid fibrils, making the Hsp40-Hsp70 machinery an attractive target for understanding the molecular basis of chaperone-mediated neuroprotection. Although functional cooperation between Hsp40 and Hsp70 is well stablished, the structural determinants governing their individual interactions with α-Synuclein remain incompletely understood. In this study, solution nuclear magnetic resonance (NMR) spectroscopy was employed to characterize the pairwise interactions between α-Synuclein, Hsp40, and Hsp70 at residue-level resolution. Uniformly ¹⁵N-labeled proteins were analyzed by two-dimensional ¹H-¹⁵N HSQC during titrations with their unlabeled binding partners. Independent titrations experiments were performed for each protein pair, allowing the interaction network to be dissected before investigating the complete chaperone system. Chemical shift perturbation (CSP) and normalized peak intensity ratios were used to identify residues affected upon complex formation and evaluate changes in local dynamics and exchange processes. These complementary NMR parameters enabled the identification of interactions interfaces and provided insight into the regime of binding. The pairwise interaction maps reveal distinct but partially overlapping recognition patterns among Hsp40, Hsp70, and α-Synuclein, indicating that each chaperone recognizes specific structural and dynamic features of the client protein while preserving the cooperative nature of the chaperone network. The observed residue-specific perturbations support a model in which transient, dynamic interactions predominate, consistent with the functional mechanism of molecular chaperones. These findings provide a structural framework for understanding the basis for future studies of the complete ternary complex and its role in preventing pathological protein aggregation.

This work was supported by Conselho Nac. Des. Cient. Tecnologico (CNPq) and FAPESP.