Inhibition of γ-secretase activity represents a potential therapeutic strategy for Alzheimer’s disease (AD). MRK-560 is a selective inhibitor with higher potency for Presenilin 1 (PS1) than for PS2, the two isoforms of the catalytic subunit of γ-secretase, although the underlying mechanism remains elusive. Here we report the cryo-electron microscopy (cryo-EM) structures of PS1 and PS2-containing γ-secretase complexes with and without MRK-560 at overall resolutions of 2.9-3.4 Å. MRK-560 occupies the substrate binding site of PS1, but is invisible in PS2. Structural comparison identifies Thr281 and Leu282 in PS1 to be the determinant for isoform-dependent sensitivity to MRK-560, which is confirmed by swapping experiment between PS1 and PS2. By revealing the mechanism for isoform-selective inhibition of presenilin, our work may facilitate future drug discovery targeting γ-secretase.

Significance γ-secretase activating protein (GSAP) has emerged as a key regulator of γ-secretase. In cells, GSAP exists primarily in the form of a 16-kDa fragment known as GSAP-16K. In this study, we report the finding that GSAP-16K undergoes phase separation in vitro and in cells. Importantly, the outcome of GSAP-16K phase separation directly regulates the protease activity of human γ-secretase. Through direct interaction with the substrate amyloid precursor protein–C-terminal 99-residue fragment, GSAP-16K in dilute phase favors the production of β-amyloid peptide 42 (Aβ42) but not Aβ40. These observations not only explain how GSAP activates γ-secretase but also identify their interaction as a target of potential therapeutic intervention.