Deducing the transmembrane domain organization of presenilin-1 in gamma-secretase by cysteine disulfide cross-linking.

Accumulation of the amyloid β-protein (Aβ) is one of the defining pathological features of Alzheimer’s disease. Aβ is produced from the amyloid β-protein precursor (APP) as a result of sequential proteolytic cleavages first by β-secretase (1) and then by γ-secretase (2). γ-Secretase is an aspartyl transmembrane protease that cleaves a number of type I membrane protein substrates, including APP and Notch, in the middle of their transmembrane domains in a poorly understood process of hydrolysis within a hydrophobic environment (3). γ-Secretase is composed of four transmembrane proteins, Aph-1, Pen-2, Nicastrin (NCT) (4–10) and presenilin (PS), which is ostensibly the catalytic component of an unusual aspartyl protease (3, 11, 12). Despite substantial progress in establishing the full identity of γ-secretase (7–9, 13, 14) and the step-wise assembly of the γ-secretase complex (7, 15, 16), the molecular structure of the protease complex or even any of its individual components remains unknown. PS, as the catalytic component, is of major interest, but only some indirect evidence about its structural arrangement has been reported to date. Indeed, it has only very recently been established that PS1 is a 9-TMD protein (17, 18). PS is endoproteolytically processed into an N-terminal fragment (NTF) and C-terminal fragment (CTF) (19). These fragments are metabolically stable, remain associated, and their formation is tightly regulated (20). Also, it is widely accepted, although not definitively proven, that the conserved aspartates D257 and D385 of presenilin constitute a catalytic dyad, and therefore these residues are expected to be proximal (21). This view implies that TMD6 and TMD7 of PS-1 should be directly adjacent to one another. Studies by Annaert et al provided evidence for a proposed “ring structure” for PS1, in which TMD1 is proximal to the C-terminus of the protein (22). Additionally, we have recently reported evidence that residue L166 in TMD3 may be a part of the active site, proposing that TMD3 is possibly in proximity to TMD6 and TMD7 (23). Finally, a report by Brunkan et al (24) provided evidence suggesting that TMD1 contributes to the active site. An understanding of the mechanism of γ-secretase proteolysis requires a detailed description of the three-dimensional organization of its transmembrane domains. In light of inherent difficulties in obtaining a structure of γ-secretase using methods such NMR spectroscopy, x-ray or electron diffraction, it is crucial to explore alternative approaches that would allow the determination of transmembrane domain organization and offer hints to how familial Alzheimer-causing mutations in PS, which are scattered along the whole sequence of the protein, cause essentially the same effect on APP processing (increasing the proportion of 42-residue Aβ to its 40-residue form). One very promising strategy, which surprisingly has not been reported in the study of γ-secretase, is cysteine mutagenesis/disulfide crosslinking. This method involves the introduction of cysteine residues at specific positions within transmembrane domains by site-directed mutagenesis and subsequent use of chemical reactivity of the introduced sulfhydryl groups. Because transmembrane cysteines are typically not accessible to modifying reagents, the most commonly used approach is cysteine-cysteine disulfide crosslinking, in which an oxidizing agent is used to catalyze the formation of disulfide bonds between proximal transmembrane cysteines and yield unique high-molecular weight products. This approach has been widely applied to deduce the oligomeric state of transmembrane proteins (25, 26), the identification of transmembrane domain organization (27–30), identification of contact points within the protein (31) and even accessing the mechanistic details of drug and substrate interaction with transmembrane targets (32). Here, we report findings about the PS structure obtained by utilizing the powerful approach of cysteine mutagenesis/disulfide crosslinking. To provide direct biochemical evidence for the arrangement of transmembrane domains in presenilin and to establish the means of probing its structure and mechanism, we initiated studies involving oxidative disulfide crosslinking of PS1.