Structure and mechanism of the γ-secretase intramembrane protease complex

Abstract

γ-Secretase is a membrane protein complex that proteolyzes within the transmembrane domain of >100 substrates, including those derived from the amyloid precursor protein and the Notch family of cell surface receptors. The nine-transmembrane presenilin is the catalytic component of this aspartyl protease complex that carries out hydrolysis in the lipid bilayer. Advances in cryoelectron microscopy have led to the elucidation of the structure of the γ-secretase complex at atomic resolution. Recently, structures of the enzyme have been determined with bound APP- or Notch-derived substrates, providing insight into the nature of substrate recognition and processing. Molecular dynamics simulations of substrate-bound enzymes suggest dynamic mechanisms of intramembrane proteolysis. Structures of the enzyme bound to small-molecule inhibitors and modulators have also been solved, setting the stage for rational structure-based drug discovery targeting γ-secretase.

Figure 1.

(A) Components of the γ-secretase complex and their membrane topology. Presenilin is a nine-TMD protein and the catalytic component of the aspartyl protease complex. Active site aspartic acids denoted in red. Upon assembly with the other three members of the complex (Nicastrin, Aph-1 and Pen-2), presenilin undergoes autoproteolysis to N-terminal fragment (NTF) and C-terminal fragment (CTF) to become the active protease complex. (B) Structure of the γ-secretase complex as determined by cryoelectron microscopy (PDB: 5A63). In this structure, TM2 was not visible, and the active site appears open (active site aspartic acids D257 and D385 in PSEN1 shown in red).

Figure 2.

(A) Structure of the γ-secretase complex bound with APP-derived substrate (PDB: 6IYC). Colors of γ-secretase components as in Fig. 1; APP substrate in orange. Solving the structure required disabling the active site through PSEN1 D385A mutation and crosslinking substrate to PSEN1 by cysteine mutagenesis and disulfide bond formation. Note that the N-terminal part of the APP TMD is in a helical conformation and enveloped by presenilin. (B) Close-up of the active site (not actually active due to D385A mutation) showing unwinding of the C-terminal part of the APP TMD, with β-sheet formation with presenilin.

Figure 3.

(A) Structure of the γ-secretase complex bound to transition-state analog inhibitor L685,458 and to modulator E2012 (PDB: 7D8X). Colors of γ-secretase components as in Figs. 1 and 2. Bound compounds are sticks colored by atom type. Note that L685,458 binds near the catalytic aspartic acids, while E2012 binds distal to the active site. (B) Close-up of L685,458 binding. The transition-state mimicking hydroxyl group of the inhibitor is coordinated with the two catalytic aspartic acids. (C) E2012 is nestled in a binding site that becomes available upon L685,458 binding to the active site. The modulator binding site includes presenilin Loop 1 residues F105 and Y106 and TM3 residues F176, F177 and I180 and Nicastrin loop residues I242 and N243.

Figure 4.

(A) Computational model of γ-secretase that is embedded in a lipid bilayer and solvated in 0.15 M NaCl aqueous solution. (B) All-atom GaMD simulations captured spontaneous activation of γ-secretase, during which the enzyme active site was poised for proteolysis of the APP substrate at the ε cleavage site. (C) Active (wildtype) and (D) shifted active (M51F) conformational states of the APP substrate-bound γ-secretase. Distinct APP intracellular domain (AICD) products were generated from the wildtype and M51F mutant APP.