Chaperone-mediated autophagy substrates bind to lid domain of Hsc70: Exploration using integrated approach

Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway crucial for cellular homeostasis. Its dysfunction is linked to cancer and neurodegenerative disorders. Hsc70 mediates CMA selectivity by recognizing proteins with ′KFERQ-type′ sequences, but the structural basis for substrate recognition and the substrate-binding mechanism of Hsc70 remain unclear. This study integrates literature, bioinformatics, AlphaFold 3 predictions, and 29 μs of molecular dynamics (MD) simulations to elucidate the lid domain interactions of Hsc70 with fifteen verified CMA substrate segments. Structural and thermodynamic residue-level analyses suggest binding occur in a hydrophobic cavity formed by alpha helix α1 and α3 in the lid domain of hsc70, with complex stabilization primarily driven by hydrophobic residues within ′KFERQ-type′ motif and its flanking regions. The robustness of our findings is confirmed by consistent convergence across force fields, substrates, and supporting literature, all identifying the same site. This study offers insights into therapeutic modulation of the CMA pathway.