Recognition of the amyloid precursor protein by human γ-secretase

The machinery behind amyloid peptides β-Amyloid peptides, which are derived from amyloid precursor protein (APP), form the plaques in the brain that are characteristic of Alzheimer's disease. Zhou et al. report a high-resolution structure of a transmembrane segment of APP bound to human γ-secretase, the transmembrane protease that cleaves APP to give β-amyloid peptides (see the Perspective by Lichtenthaler and Güner). Disease-associated mutations within presenilin-1, the catalytic subunit of APP, likely affect how the substrate is bound and thus which peptides are generated, with some being more amyloidogenic. It may now be possible to exploit the features of substrate binding to design inhibitors. Science, this issue p. eaaw0930; see also p. 690 A view of how the substrate is recognized facilitates understanding of Alzheimer’s disease–associated mutations in γ-secretase. INTRODUCTION Alzheimer’s disease (AD) is characterized by amyloid plaques in the brains of patients. The primary components of amyloid plaques are β-amyloid peptides (Aβs), which are derived from the amyloid precursor protein (APP). APP is first cleaved by α- or β-secretase to generate an 83- or 99-residue transmembrane (TM) fragment (APP-C83 or APP-C99), respectively. APP-C99 is then cleaved by the intramembrane aspartyl protease γ-secretase to generate the peptides Aβ48, Aβ45, Aβ42, and Aβ38 or Aβ49, Aβ46, Aβ43, and Aβ40. Of these, Aβ42 and Aβ43 are particularly prone to aggregation and formation of the amyloid plaques. Another substrate of γ-secretase is the Notch receptor. Aβ oligomers may contribute to AD development. Therefore, inhibition of γ-secretase represents a potential therapeutic treatment for AD. Unfortunately, γ-secretase inhibitors caused severe side effects without any clear clinical benefits for AD patients, perhaps owing to their inhibition of Notch cleavage. Human γ-secretase comprises four subunits: presenilin (PS), PEN-2, APH-1, and nicastrin. As the catalytic subunit of γ-secretase, presenilin has two isoforms (PS1 and PS2). PS1—and, to a lesser extent, PS2 and APP—are frequently targeted for mutations in familial AD patients. Although free γ-secretase has been structurally characterized, how it recognizes APP remains largely unknown. RATIONALE Structural comparison of APP and Notch recognition by γ-secretase may reveal differences that can be exploited toward the design of substrate-specific inhibitors. However, the γ-secretase–substrate complex is extremely transient and has defied all efforts of isolation for structural studies. To this end, we developed a cross-linking strategy that involves mutation of two specific residues to Cys and thus allows formation of a disulfide bond between PS1 and the substrate. Using this approach, we obtained a cross-linked complex between a variant of human γ-secretase [with PS1-Q112C (Gln112→Cys)] and APP-C83 (V695C). To avoid substrate cleavage, the catalytic residue Asp385 in PS1 was mutated to Ala. Because PS1 undergoes autoproteolysis during γ-secretase assembly to produce an N-terminal fragment (NTF) and a C-terminal fragment (CTF), these two fragments of PS1 in the γ-secretase were coexpressed. The final γ-secretase contains PS1 (NTF-Q112C, CTF-D385A), PEN-2, APH-1aL (a specific isoform of APH-1), and nicastrin. This γ-secretase was cross-linked to APP-C83 (V695C), and the complex was analyzed by cryo–electron microscopy (cryo-EM). RESULTS The cryo-EM structure of the cross-linked human γ-secretase–APP-C83 complex was determined at an average resolution of 2.6 Å. The quality of the EM map allows unambiguous identification of the bound APP fragment, which traverses through the center of the γ-secretase TM domain. Compared to substrate-free γ-secretase, the flexible transmembrane helix 2 (TM2) of PS1 becomes ordered upon binding to APP-C83 and contributes to its recognition, and the C-terminal portion of TM6 of PS1 is unraveled into a rigid loop followed by a short α helix (designated as TM6a). The TM of APP closely interacts with five surrounding TMs (TM2, TM3, TM5, TM6, and TM7) of PS1. Notably, the APP sequences on the C-terminal side of the TM form a β strand, which, together with two APP-induced β strands of PS1, constitutes a hybrid β sheet on the intracellular side. This β sheet guides γ-secretase to the scissile peptide bond of APP just preceding the N terminus of the β strand. Mutations that compromise the hybrid β sheet result in abrogation of APP cleavage by γ-secretase. Notably, residues at the interface between PS1 and APP are heavily targeted by recurring AD mutations. CONCLUSION The structure of human γ-secretase bound to APP-C83 constitutes a framework for understanding the function and disease relevance of γ-secretase. This structure, together with that of γ-secretase bound to Notch, reveals contrasting features of substrate recognition, which may be applied toward the design of substrate-specific inhibitors. The cryo-EM structure of human γ-secretase bound to APP at 2.6-Å resolution. (Left) Overall structure of the γ-secretase–APP-C83 complex. PS1, cyan; PEN-2, yellow; APH-1, pink; nicastrin (NCT), green; APP, blue. (Top right) Close-up view of the hybrid β sheet. The three-stranded β sheet comprises a β strand from APP and two β strands from the extended loop sequence between the NTF and CTF of PS1. (Bottom right) Close-up structural comparison between free APP (orange) and APP bound to γ-secretase (blue). The two initial cleavage sites of γ-secretase are marked by arrows. Cleavage of amyloid precursor protein (APP) by the intramembrane protease γ-secretase is linked to Alzheimer’s disease (AD). We report an atomic structure of human γ-secretase in complex with a transmembrane (TM) APP fragment at 2.6-angstrom resolution. The TM helix of APP closely interacts with five surrounding TMs of PS1 (the catalytic subunit of γ-secretase). A hybrid β sheet, which is formed by a β strand from APP and two β strands from PS1, guides γ-secretase to the scissile peptide bond of APP between its TM and β strand. Residues at the interface between PS1 and APP are heavily targeted by recurring mutations from AD patients. This structure, together with that of γ-secretase bound to Notch, reveal contrasting features of substrate binding, which may be applied toward the design of substrate-specific inhibitors.