Investigation of CMA Substrate Interactions with Hsc70 Reveals Potential Auxiliary Binding Sites

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 44 s of molecular dynamics (MD) simulations to elucidate the lid domain interactions of Hsc70 with fifteen experimentally verified CMA substrate segments. Structural and thermodynamic residue-level analyses suggest stable binding of substrates in two distinct sites. The first site resides in a hydrophobic cavity formed by alpha helix 1 and 3 in the lid domain of Hsc70, with complex stabilization primarily driven by hydrophobic residues within KFERQ-type motif and its flanking regions. The second stable site was observed to be canonical hydrophobic cleft in Hsc70, with complex stabilization primarily driven by hydrophobic residues within KFERQ-type motif as well as the through water-mediated interactions by the lid domain. The robustness of our findings is confirmed by consistent convergence across force fields, substrates, and supporting literature, all identifying the same site. We propose these sites may facilitate upstream or downstream recognition and activation events. Author SummaryCells must constantly remove damaged or unnecessary proteins in order to stay healthy. One important pathway that performs this task is chaperone-mediated autophagy. When this pathway fails, it has been linked to conditions such as cancer and neurodegenerative diseases. A key player in this process is the protein Hsc70, which recognizes short sequence signatures in target proteins and directs them for degradation. However, how Hsc70 physically recognizes these signatures has remained poorly understood. In this study, we investigated how Hsc70 interacts with a range of confirmed target protein segments. We combined information from prior experimental studies, with computational structure prediction and extensive molecular simulations to explore these interactions in detail. Our results indicate that Hsc70 can bind target proteins at two distinct regions, each stabilized mainly by hydrophobic amino acids within and/or around the recognition signal, with additional contribution from water-mediated interactions in the latter site. Notably, these binding modes were consistent across different targets and computational conditions. Together, our findings provide a clearer structural picture of how selectivity is achieved in this degradation pathway. This improved understanding may help guide future studies on how chaperone-mediated autophagy is regulated and how it becomes disrupted in disease.