␥-Secretase is a structurally enigmatic multiprotein complex that catalyzes intramembrane proteolysis of a variety of substrates, including the amyloid ␤-protein precursor of Alzheimer's disease and the Notch receptor essential to cell differentiation. The active site of this transmembrane aspartyl protease apparently lies at the interface between two subunits of presenilin-1 (PS1); however, evidence suggests the existence of an initial substrate-binding site that is distinct from the active site. Here, we report that photoaffinity probes based on potent helical peptide inhibitors and designed to mimic the amyloid ␤-protein precursor substrate bind specifically to the PS subunit interface, at a site close to the active site. The location of the helical peptide-binding site suggests that substrate passes between the two PS1 subunits to access the active site. An aggressive Alzheimer-causing mutation in PS1 strongly reduced photolabeling by a transition-state analogue but not by helical peptides, providing biochemical evidence that the pathological effect of this PS mutation is due to alteration of the active-site topography.affinity labeling ͉ mechanism ͉ membrane protein ͉ protease T he ␥-secretase complex is ostensibly a multicomponent aspartyl protease with presenilin (PS) as the catalytic component (1-3). Three other integral membrane proteins, nicastrin (NCT), Aph-1, and Pen-2, are necessary and, along with PS, sufficient members of the protease complex (4-10). However, despite the elucidation of the identity of ␥-secretase, its structure and mechanism remain uncertain. ␥-Secretase cleaves amide bonds within the transmembrane regions of its substrates, a poorly understood process of hydrolysis within a hydrophobic environment (3). The study of transition-state analogue inhibitors of ␥-secretase led to the suggestion that ␥-secretase is an aspartyl protease and that two conserved aspartates in PS are catalytic residues (11-13). PS is processed into an N-terminal fragment (NTF) and C-terminal fragment (CTF). These fragments are metabolically stable and remain associated, and their formation is tightly regulated (14). The direct binding of transition-state analogue ␥-secretase inhibitors to these fragments strongly suggested that the active site is at the NTF͞CTF heterodimeric interface (15, 16), consistent with the fact that each subunit contributes one of the two critical aspartates (13).Because of its requirement for water, the active site of ␥-secretase is thought to be in the protein interior to avoid the hydrophobic environment of the lipid bilayer (17). As a result, the integral membrane substrates initially should interact on the surface of the protease before entering the internal active site. In fact, an endogenous ␥-secretase substrate copurifies with the protease complex isolated from an immobilized transition-state analogue (10, 18), suggesting that substrate can bind at some other site while the immobilized inhibitor occupies the active site. Such substrate-binding sites that are distinct from the...

␥-Secretase is a protease complex of four integral membrane proteins, with presenilin (PS) as the apparent catalytic component, and this enzyme processes the transmembrane domains of a variety of substrates, including the amyloid ␤-protein precursor and the Notch receptor. Here we explore the mechanisms of structurally diverse ␥-secretase inhibitors by examining their ability to displace an active site-directed photoprobe from PS heterodimers. Most ␥-secretase inhibitors, including a potent inhibitor of the PS-like signal peptide peptidase, blocked the photoprobe from binding to PS1, indicating that these compounds either bind directly to the active site or alter it through an allosteric interaction. Conversely, some reported inhibitors failed to displace this interaction, demonstrating that these compounds do not interfere with the protease by affecting its active site. Differential effects of the inhibitors with respect to photoprobe displacement and in cell-based and cell-free assays suggest that these compounds are important mechanistic tools for deciphering the workings of this intramembrane-cleaving protease complex and its similarity to other polytopic aspartyl proteases.Cerebral accumulation of the amyloid-␤ protein (A␤) 1 is considered a central event in the pathogenesis of Alzheimer's disease (AD). A␤ is produced via ␤-and ␥-secretases, proteases that have become important therapeutic targets for AD (1). ␥-Secretase plays a crucial role in determining the proportion of two forms of A␤, A␤ 40 and A␤ 42 . The 42-residue A␤ 42 is more prone to fibril formation and is disproportionately present in the plaques characteristic of the AD brain. Accumulating evidence (2-4) strongly suggests that ␥-secretase is an intramembrane-cleaving aspartyl protease with presenilin (PS) as the catalytic component. Three other multipass membrane proteins, nicastrin, Aph-1, and Pen-2, are genetically linked to ␥-secretase activity (5-7), and biochemical isolation has provided evidence that these proteins are indeed necessary members of the protease complex (8 -10). Despite the remarkable progress in uncovering the identity of ␥-secretase, its mechanism of action remains unclear.A body of work (11) suggests that ␥-secretase cleaves amide bonds within the transmembrane regions of its substrates, a poorly understood process of hydrolysis within a hydrophobic environment. Elucidating the molecular interaction between an inhibitor and its enzyme target can help identify the enzyme and provide insight into the catalytic mechanism. The study of peptidomimetic inhibitors of ␥-secretase that contain classic aspartyl protease transition state-mimicking moieties led to the suggestion that ␥-secretase is an aspartyl protease and that two conserved aspartates in presenilins are catalytic residues (12). PS is processed into N-terminal (NTF) and C-terminal (CTF) fragments. These fragments are metabolically stable, remain associated, and their formation is tightly regulated, suggesting that together they are the bioactive form of PS (12). Th...

Signaling from the Notch (N) receptor is essential for proper cell-fate determinations and tissue patterning in all metazoans. N signaling requires a presenilin (PS)-dependent transmembrane-cleaving activity that is closely related or identical to the gamma-secretase proteolysis of the amyloid-beta precursor protein (APP) involved in Alzheimer's disease pathogenesis. Here, we show that N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester, a potent gamma-secretase inhibitor reported to reduce amyloid-beta levels in transgenic mice, prevents N processing, translocation, and signaling in cell culture. This compound also induces developmental defects in Drosophila remarkably similar to those caused by genetic reduction of N. The appearance of this phenocopy depends on the timing and dose of compound exposure, and effects on N-dependent signaling molecules established its biochemical mechanism of action in vivo. Other gamma-secretase inhibitors caused similar effects. Thus, the three-dimensional structure of the drug-binding site(s) in Drosophila gamma-secretase is remarkably conserved vis-à-vis the same site(s) in the mammalian enzyme. These results show that genetics and developmental biology can help elucidate the in vivo site of action of pharmacological agents and suggest that organisms such as Drosophila may be used as simple models for in vivo prescreening of drug candidates.