Understanding the molecular mechanisms controlling the physiological and pathological activity of γ-secretase represents a challenging task in Alzheimers disease (AD) research. The assembly and proteolytic activity of this enzyme require the correct interaction of the 19 transmembrane domains (TMDs) present in its four subunits, including Presenilin (PS1 or PS2), the γ-secretase catalytic core. GxxxG and GxxxG-like motifs are critical for TMDs interactions as well as for protein folding and assembly. The GxxxG motifs on γ-secretase subunits (e.g. APH-1) or on γ-secretase substrates (e.g. APP) are known to be involved in γ-secretase assembly and in Aβ peptide production, respectively. We identified on PS1 and PS2 TMD8 two highly conserved AxxxAxxxG motifs. The presence of a mutation causing inherited form of AD (FAD) in the PS1 motif suggested their involvement in the physiopathological configuration of the γ-secretase complex. In this study, we targeted the role of these motifs on TMD8 of PSs, focusing on their role in PS assembly and catalytic activity. Each motif was mutated and the impact on complex assembly, activity, as well as substrate docking was monitored. Different amino acid substitutions on the same motif resulted in opposite effects on γ-secretase activity, without affecting the assembly or significantly impairing the maturation of the complex. Our data suggests that AxxxAxxxG motifs in PS TMD8 are key determinants for the conformation of the mature γ-secretase complex, participating to the switch between the physiological and pathological functional conformations of the γ-secretase.