Mechanistic Insights into How Juxtamembrane Residue Modulation Leads To LAMP2A Inactivation in Chaperone-Mediated Autophagy.

The lysosome-associated membrane protein type 2A (LAMP2A) is a critical mediator of chaperone-mediated autophagy (CMA), a selective degradation pathway essential for cellular homeostasis. The trimeric assembly of LAMP2A serves as an intermediate in forming higher-order oligomers that are responsible for substrate translocation and degradation. However, the molecular determinants of the trimeric stability remain poorly understood. We performed 30 μs of all-atom molecular dynamics simulations in a realistic lysosomal membrane environment to investigate how different protonation states of juxtamembrane histidine residues influence the structural dynamics of wild-type (WT) LAMP2A and compare these with an experimentally characterized inactive mutant, in which four charged residues were replaced by alanines. Comparative analyses reveal that in the WT protein, this region is stabilized through charged lipid nanoclusters and contributes to maintaining proper tilt angles and membrane anchoring of the monomeric and trimeric assemblies, while the mutant buries these residues within the lipid bilayer due to hydrophobic mismatch, leading to altered tilting, reduced dynamicity, disrupted oligomeric stability, altered lipid distribution, and membrane properties. We identified key interacting residues stabilizing the WT oligomeric state and demonstrated how their loss compromises trimeric assembly in the mutant. Our results offer mechanistic insights into how the disruption of juxtamembrane residues impairs CMA activity, with potential implications for CMA regulation.