Structure of the catalytic pore of gamma-secretase probed by the accessibility of substituted cysteines - PubMed (original) (raw)

Structure of the catalytic pore of gamma-secretase probed by the accessibility of substituted cysteines

Chihiro Sato et al. J Neurosci. 2006.

Abstract

Several single-span membrane proteins are cleaved within their transmembrane domains (TMDs) by intramembrane-cleaving proteases, although the structure of the active site executing intramembrane cleavage remains unknown. Here we use the substituted cysteine accessibility method to examine the structure of presenilin-1, a catalytic subunit of gamma-secretase, involved in amyloid beta protein generation in Alzheimer's disease and Notch signaling. We show that TMD6 and TMD7 of presenilin-1 contribute to the formation of a hydrophilic pore within the membrane. Residues at the luminal portion of TMD6 are predicted to form a subsite for substrate or inhibitor binding on the alpha-helix facing a hydrophilic milieu, whereas those around the GxGD catalytic motif within TMD7 are highly water accessible, suggesting formation of a hydrophilic structure within the pore. Collectively, our data suggest that the active site of gamma-secretase resides in a catalytic pore filled with water within the lipid bilayer and is tapered around the catalytic aspartates.

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Figures

Figure 1.

Figure 1.

Locations of the PS1 cysteine mutations used in this study. A schematic depiction of human PS1 based on a 9 TMD topology is shown. Catalytic aspartates are shown by yellow stars. Endogenous cysteines replaced with serine in PS1/Cys(−) are indicated by black circles. Amino acid residues substituted to cysteines are shown by a circle with a single-letter character representing the original amino acid. Mutated cysteines that retained γ-secretase activity and thus were analyzed as controls are indicated as green circles. Single-Cys mt PS1 in HR6 and HR8 analyzed in this study are indicated by purple and pink circles, respectively. Cysteine substitutions that resulted in a loss of γ-secretase activity are indicated by gray circles.

Figure 2.

Figure 2.

SCAM analysis of single-Cys mt PS1 at representative positions. A, Western blot analysis of single-Cys mt PS1 stably expressed in DKO cells. Endoproteolysis of PS1 and restoration of the expression of mature glycosylated Nct and Pen-2 in all single-Cys mt PS1 are shown. B, Effect of single-Cys mt PS1 on Aβ levels secreted from DKO cells coexpressing APPNL. Error bars indicate mean ± SE (n = 3). C, Biotin-labeling experiment using MTSEA-biotin in intact cells (left) and microsomes (right). D, Labeling competition by preincubation with MTSES and MTSET. MEF, Mouse embryonic fibroblast; wt, wild type.

Figure 3.

Figure 3.

SCAM analysis of single-Cys mt PS1 in HR6. A, Biotin-labeling experiment using MTSEA-biotin in intact cells (top) and microsomes (middle). The amount of PS1 NTF in the input fraction is shown in the bottom panel. B, Labeling competition by MTSES and MTSET.

Figure 4.

Figure 4.

SCAM analysis of single-Cys mt PS1 around HR8. A, Biotin-labeling experiment using MTSEA-biotin in intact cells (top) and microsomes (middle). The amount of PS1 CTF in the input fraction is shown in the bottom panel. B, C, Biotin labeling of single-Cys mt PS1 around HR8 was performed after preincubation with MTSES or MTSET in intact cells (B) or microsomes (C). Locations of cysteine mutations are shown on the left side of the panel.

Figure 5.

Figure 5.

Labeling competition by γ-secretase inhibitors. Biotin labeling of single-Cys mt PS1 was conducted after preincubation with L-685,458, pep15, or DAPT in intact cells (A) or microsomes (B). Locations and predicted topology of cysteine mutations are shown on the left and right sides of the panel, respectively.

Figure 6.

Figure 6.

Cross-linking experiment using MTS cross-linkers. Thiol-mediated cross-linking experiments of single-Cys or double-Cys mt in TMD6 (L250C) and TMD7 (L383C and I387C) using MTS cross-linkers are shown.

Figure 7.

Figure 7.

Hypothetical structure around the catalytic site of γ-secretase. A, A summary of SCAM analysis. Catalytic aspartates are indicated by yellow stars. Cysteine mutants that were labeled by MTSEA-biotin are shown by a white letter in a black circle. The labeling of residues that were effectively competed by MTSES or MTSET is shown in a blue frame, and the unaffected residues are in a green frame. Residues that were not labeled by MTSEA-biotin or unanalyzed are shown by a black letter in a white and gray circle, respectively. Putative locations of L-685,458 and DAPT within the catalytic pore of PS1 are indicated by orange and pink lines, respectively. B, α-Helical wheel alignment consisting of the 18 amino acids of TMD6 of human PS1 starting at the N-terminal tryptophan 244. Catalytic aspartate at position 257 is indicated by a star. Residues that were biotin labeled and competed by L-685,458 (i.e., A246 and L250) are shown by orange circles. A260 was indicated by a green circle. Note that A246 and L250 are aligned on the same interface with D257 in an α-helical model. C, Schematic depiction of the configuration of the catalytic pore. TMD6 and TMD7 are shown in purple and pink lines, respectively. Catalytic aspartates are shown in yellow stars. Regions that serve as subsites, including the conserved motif forming a catalytic site (pink oval), are indicated in orange. Residues that are close to the aspartates but putatively facing a geometrically different hydrophilic environment from the catalytic pore are shown in green.

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