Modulating hinge flexibility in the APP transmembrane domain alters γ-secretase cleavage

Intramembrane cleavage of the {beta}-amyloid precursor protein C99 substrate by {gamma}-secretase is implicated in Alzheimers disease pathogenesis. Since conformational flexibility of a di-glycine hinge in the C99 transmembrane domain (TMD) might be critical for {gamma}-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline. CD, NMR and hydrogen/deuterium exchange measurements as well as MD simulations showed that the mutations distinctly altered the intrinsic structural and dynamical properties of the TMD. However, although helix destabilization/unfolding was not observed at the initial {varepsilon}-cleavage sites of C99, both mutants impaired {gamma}-secretase cleavage and altered its cleavage specificity. Moreover, helix flexibility enabled by the di-glycine hinge translated to motions of other helix parts. Our data suggest that both local helix stabilization and destabilization in the di-glycine hinge may decrease the occurrence of enzyme-substrate complex conformations required for normal catalysis and that hinge mobility can be conducive for productive substrate-enzyme interactions.