Capture of Amyloid Precursor Protein Fragments by an Engineered Water-Soluble γ-Secretase Variant

The Amyloid Precursor Protein (APP) fragments involve plaque formation when generated by γ-secretase. Mechanistic interrogation of γ-secretase substrate selection has been historically constrained by the hydrophobic and detergent-sensitive nature of its transmembrane core. Here, we generate the first fully water-soluble QTY-engineered analogue of the γ-secretase transmembrane scaffold and evaluate its ability to bind the APP transmembrane helix across 250ns molecular dynamics simulations. The QTY-code engineered protein preserved helical topology, orientation, and packing with 1.7 angstrom precision. MM-GBSA analyses yielded a mean binding free energy of -173.1±24.9 kcal/mol, supported by residue-level hotspot contributions and APP positions Arg61, Lys65, and Phe78. We further uncovered a dense cluster of co-evolving residues at the interface between the Presenilin-1 substrate-binding pocket and the flexible loop region of APP. These findings demonstrate that substrate recognition is preserved outside a lipid environment, establishing QTY-solubilized γ-secretase as a powerful platform for mechanistic dissection, mutational analysis, and biosensor development.