Structural and Chemical Biology of Presenilin Complexes

Abstract

The presenilin proteins are the catalytic subunits of a tetrameric complex containing presenilin 1 or 2, anterior pharynx defective 1 (APH1), nicastrin, and PEN-2. Other components such as TMP21 may exist in a subset of specialized complexes. The presenilin complex is the founding member of a unique class of aspartyl proteases that catalyze the γ, ɛ, ζ site cleavage of the transmembrane domains of Type I membrane proteins including amyloid precursor protein (APP) and Notch. Here, we detail the structural and chemical biology of this unusual enzyme. Taken together, these studies suggest that the complex exists in several conformations, and subtle long-range (allosteric) shifts in the conformation of the complex underpin substrate access to the catalytic site and the mechanism of action for allosteric inhibitors and modulators. Understanding the mechanics of these shifts will facilitate the design of γ-secretase modulator (GSM) compounds that modulate the relative efficiency of γ, ɛ, ζ site cleavage and/or substrate specificity.

Figure 1.

A diagrammatic representation of the presenilin-complex subunit component proteins including presenilin, PEN-2, anterior pharynx defective 1 (APH1), and nicastrin. The orientations of the membrane proteins relative to the cytoplasmic and lumenal faces of the membrane are depicted. Presenilin has nine predicted transmembrane (TM) domains, PEN-2 has two partial TM domains and a final fully spanning TM domain, APH1 has seven, and nicastrin has one TM domain.

Figure 2.

A diagrammatic representation of the intramembranous cleavage sites within the transmembrane (TM) domain of substrate proteins is shown using the APP-C100 substrate released from full-length APP by prior β-secretase cleavage as the canonical substrate. The predominant and minor series of cleavages are depicted. The N-terminal fragment forms the amyloid-β (Aβ) peptide associated with neurotoxicity in Alzheimer’s disease (AD). The C-terminal fragment is a putative signaling molecule that can be translocated to the nucleus. The two smaller internal fragments are rapidly degraded.

Figure 3.

Single-particle electron density maps of the human presenilin complex. (A) The initial low resolution (17 Å) map (left) and the subsequent higher resolution cryo-electron microscopy (cryo-EM) map (3.4 Å) show the essential features of the structure, with the nicastrin ectodomain in the smaller head and the transmembrane (TM) domains embedded in the larger base domain. Both structures show the existence of a lateral cleft, which opens into a central horseshoe-shaped cavity. (B) A 180° rotation of the model in A shows the relative positions of the two aspartate residues on TM6 and TM7. (C) Superimposition of the maps of the compound E–bound presenilin complex at 17 Å and the similar DAPT-bound complex at 3.4-Å resolution (left). Both show evidence of subtle conformational changes with movement of TM domains that result in closure of the substrate-binding pocket. In the higher resolution cryo-EM model (right), a conformational change at the end of TM6 can be visualized (red arrow) following DAPT (red spheres) binding, which likely alters acquisition and transfer of substrate to the active site.

Figure 4.

Structure of γ-secretase inhibitor compounds. (A) Substrate-based peptidomimetic inhibitors and probes. (B) L458 and photoprobe analogs used for photophore walking. (C) Biotinylated L458 probes with different linker lengths. (D) Clickable L458 and CBAP probes. (E) Difluoro ketone compounds and hydroxyethyl urea peptidomimetics. (F) Clinical γ-secretase inhibitors for Alzheimer's disease and cancer.

Figure 4.

Structure of γ-secretase inhibitor compounds. (A) Substrate-based peptidomimetic inhibitors and probes. (B) L458 and photoprobe analogs used for photophore walking. (C) Biotinylated L458 probes with different linker lengths. (D) Clickable L458 and CBAP probes. (E) Difluoro ketone compounds and hydroxyethyl urea peptidomimetics. (F) Clinical γ-secretase inhibitors for Alzheimer's disease and cancer.

Figure 4.

Structure of γ-secretase inhibitor compounds. (A) Substrate-based peptidomimetic inhibitors and probes. (B) L458 and photoprobe analogs used for photophore walking. (C) Biotinylated L458 probes with different linker lengths. (D) Clickable L458 and CBAP probes. (E) Difluoro ketone compounds and hydroxyethyl urea peptidomimetics. (F) Clinical γ-secretase inhibitors for Alzheimer's disease and cancer.

Figure 4.

Structure of γ-secretase inhibitor compounds. (A) Substrate-based peptidomimetic inhibitors and probes. (B) L458 and photoprobe analogs used for photophore walking. (C) Biotinylated L458 probes with different linker lengths. (D) Clickable L458 and CBAP probes. (E) Difluoro ketone compounds and hydroxyethyl urea peptidomimetics. (F) Clinical γ-secretase inhibitors for Alzheimer's disease and cancer.

Figure 4.

Structure of γ-secretase inhibitor compounds. (A) Substrate-based peptidomimetic inhibitors and probes. (B) L458 and photoprobe analogs used for photophore walking. (C) Biotinylated L458 probes with different linker lengths. (D) Clickable L458 and CBAP probes. (E) Difluoro ketone compounds and hydroxyethyl urea peptidomimetics. (F) Clinical γ-secretase inhibitors for Alzheimer's disease and cancer.

Figure 4.

Structure of γ-secretase inhibitor compounds. (A) Substrate-based peptidomimetic inhibitors and probes. (B) L458 and photoprobe analogs used for photophore walking. (C) Biotinylated L458 probes with different linker lengths. (D) Clickable L458 and CBAP probes. (E) Difluoro ketone compounds and hydroxyethyl urea peptidomimetics. (F) Clinical γ-secretase inhibitors for Alzheimer's disease and cancer.

Figure 5.

Allosteric γ-secretase modulators. (A) Cartoon showing allosteric interactions between the active site and the binding sites of heterocyclic and acid γ-secretase modulator compounds. (B) Structure of acid γ-secretase modulator compounds. (C) Structure of heterocyclic γ-secretase modulator compounds.

Figure 5.

Allosteric γ-secretase modulators. (A) Cartoon showing allosteric interactions between the active site and the binding sites of heterocyclic and acid γ-secretase modulator compounds. (B) Structure of acid γ-secretase modulator compounds. (C) Structure of heterocyclic γ-secretase modulator compounds.