Stabilization / destabilization of the APP transmembrane domain by mutations in the di-glycine hinge alter helical structure and dynamics, and impair cleavage by γ-secretase

γ-Secretase is a pivotal intramembrane protease that is involved in the generation of toxic Alzheimers disease-associated peptides from the β-amyloid precursor protein C-terminal fragment C99. Determinants of γ-secretase substrates are not defined yet and in the absence of recognition motifs, helix-stability and dynamics have been discussed as critical factors for recognition and cleavage of γ-secretase. Since conformational flexibility of a di-glycine hinge region in the C99 transmembrane domain (TMD), which separates the TMD into N- and C-terminal parts, might play a role in the processing of C99 by γ-secretase, we mutated one of the glycines, G38, to a helix-stabilizing leucine and to a helix-distorting proline residue. While γ-secretase cleavage of the G38L mutant was reduced, that of G38P was strongly impaired. Furthermore, cleavage precision of γ-secretase was dramatically altered by the mutations. CD and NMR spectroscopy, hydrogen/deuterium exchange measurements as well as in silico modeling by MD simulations to assess structural and dynamical parameters showed that the mutations affected the TMD helix properties, in particularly for the G38P mutant. However, helix destabilization was not observed at the ϵ-cleavage sites of C99, suggesting that local unfolding of the TMD to allow access to the scissile bonds requires the presence of the protease. Our data suggest that conformational relaxations of substrate and enzyme during substrate recruitment required for catalysis are established only when substrate and protease come into contact.