Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis
- German Center for Neurodegenerative Diseases, Germany
- Center of Functional Protein Assemblies and Physics Department T38, Technical University of Munich, Germany
- Department of Pharmacotherapeutics II, Osaka Medical and Pharmaceutical University, Japan
- Institute for Diabetes and Obesity, Monoclonal Antibody Core Facility, Helmholtz Munich, German Research Center for Environmental Health, Germany
- Biomedical Center (BMC), Division of Metabolic Biochemistry, Faculty of Medicine, Germany
Figures
Figure 1 with 1 supplement
Cleavage of APP C99 by PSH.
(A) Schematic illustration of APP C99 cleavage by PSH. PSH cleaves C99 and releases an AICD fragment and Aβ peptides. The epitope of the PSH specific antibody 6F4 in the loop between TMD6 and TMD7 …
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Figure 1—source data 1
Immunoblot images (raw and annotated) of cleavage assay (Source data for Figure 1B, C).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig1-data1-v2.zip
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Figure 1—source data 2
Calculated and observed masses for Aβ species in MALDI-TOF mass spectrometry (Source data for Figure 1D).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
MS specificity control for Aβ cleavage products generated from APP C99 by PSH.
Representative MALDI-TOF MS spectrum of PSH cleavage assay in DDM micelles in the presence of L-685,458 from four independent biological replicates. The intensity of the highest peak was set to 100%.
Figure 2 with 2 supplements
Comparison of PSH cleavage activity and processivity in DDM micelles and POPC bilayer.
(A) Analysis of PSH activity in DDM micelles and POPC vesicles after incubation with C100-His6 substrate at 37 °C overnight by immunoblotting for AICD (Y188) and Aβ (2D8). Immunoblotting of PSH …
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Figure 2—source data 1
Immunoblot images (raw and annotated) of cleavage assays (Source data for Figure 2A, B).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig2-data1-v2.zip
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Figure 2—source data 2
Calculated and observed masses for Aβ, and AICD species in MALDI-TOF mass spectrometry (Source data for Figure 2C and D).
The differences between calculated and observed masses for AICD species arise from different adducts as specified in the respective tables. The GSI controls in Figure 2—figure supplement 2B show that the observed peaks are specific for AICD species derived from PSH cleavage.
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Reconstitution of PSH in POPC vesicles.
(A) Fluorometric analysis of gel filtration fractions of PSH-containing POPC vesicles with rhodamine-DHPE as fluorescent marker lipid. A representative graph from three independent biological …
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Figure 2—figure supplement 1—source data 1
Raw values of measured fluorescence intensity (Source data for Figure 2—figure supplement 1A).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig2-figsupp1-data1-v2.xlsx
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Figure 2—figure supplement 1—source data 2
Immunoblot images (raw and annotated) of PSH reconstitution (Source data for Figure 2—figure supplement 1B).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig2-figsupp1-data2-v2.zip
Figure 2—figure supplement 2
MS specificity controls for Aβ and AICD cleavage products generated from APP C99 by PSH.
(A, B) MALDI-TOF MS analysis of Aβ (A) and AICD (B) species from PSH cleavage assays in DDM micelles and POPC vesicles in the presence of L-685–458 at pH 7.0. Representative mass spectra from four …
Figure 3
Cleavage of APP C83 by PSH.
(A) Analysis of PSH activity in DDM and POPC environment after incubation with C83-His6 and C100-His6 substrates at 37 °C and pH 7.0 by immunoblotting for AICD (penta-His) and p3 (Aβ (22-35)). …
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Figure 3—source data 1
Immunoblot images (raw and annotated) of cleavage assays (Source data for Figure 3A).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig3-data1-v2.zip
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Figure 3—source data 2
Raw values of p3 and Aβ concentrations measured in the ECL-IA and calculated p3-40/p3-42 and Aβ40/Aβ42 ratios (Source data for Figure 3B, C).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig3-data2-v2.xlsx
Figure 4 with 2 supplements
PSH homology model.
(A) Alignment of the modeled holo form (model 2) of PSH (blue) with APP C83 substrate (orange) and the crystal structure of PSH (PDB 4HYG) in the apo form (green) in side view (left panel) and top …
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Figure 4—source data 1
Immunoblot images (raw and annotated) of cleavage assays (Source data for Figure 4E).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig4-data1-v2.zip
Figure 4—figure supplement 1
Comparison of the three different homology models.
(A) Alignment of the three modeled holo forms of PSH in side view (left panel) and top view (right panel). (B) RMSD values between the available crystal structures (PDB 4HYG and 4Y6K) and the three …
Figure 4—figure supplement 2
Alignment of PS1 and PSH used for homology modeling based on the TMD annotations of the available cryo-EM or crystal structures, respectively (PDB 6IYC for PS1 and PDB 4HYG for PSH).
Active site motifs including the catalytic aspartate residues (red) are highlighted in bold. The β2-strands of γ-secretase and PSH are indicated as orange arrows and residues that were mutated in …
Figure 5 with 3 supplements
MD simulations of holo PSH forms in DDM micelle or POPC bilayer.
(A) PSH with bound C83 substrate (model 2) embedded in a DDM micelle environment (upper panel) and a POPC bilayer (lower panel). (B) The average number of H-bonds formed between the β3-strand of C83 …
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Figure 5—source data 1
Raw values of simulation data analysis (Source data for Figure 5B–E).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig5-data1-v2.zip
Figure 5—figure supplement 1
Homology modeling of the holo form PSH in complex with C83 in DDM and POPC environments.
(A) The backbone RMSD of PSH of different models in DDM (red) and POPC (blue) averaged over three trajectories. The black dashed line indicates an RMSD of 4 Å. (B) Enlarged representation of …
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Figure 5—figure supplement 1—source data 1
Raw values of simulation data analysis (Source data for Figure 5—figure supplement 1A, B).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig5-figsupp1-data1-v2.zip
Figure 5—figure supplement 2
Secondary structure of PSH TMD6a and surrounding residues over simulation time.
(A–C) Secondary structure of PSH TMD6a (orange) and surrounding residues of model 1 (A), model 2 (B), and model 3 (C) in DDM micelle (top) and POPC bilayer (bottom) environments over the simulation …
Figure 5—figure supplement 3
Comparison of models 2 and 3.
(A) Structural comparison of model 2 (blue) and 3 (gray). TMD6a is spatially closer to the C83 substrate in model 2. (B) Comparison of the backbone RMSFs of PSH model 2 (blue) and 3 (gray) in POPC …
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Figure 5—figure supplement 3—source data 1
Raw values of simulation data analysis (Source data for Figure 5—figure supplement 3B, C).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig5-figsupp3-data1-v2.zip
Figure 6 with 3 supplements
Destabilization of PSH TMD6a in a DDM micelle.
(A) Hydrophobic interactions between PSH regions and the C83 substrate. The right panel shows an enlarged view of the interaction of a hydrophobic patch (gray surface) of TMD6a with V50 and L52 of …
Figure 6—figure supplement 1
Hydrophobic interactions of PS1 TMD6a in substrate-bound and GSI-bound γ-secretases.
(A, B) Interactions between TMD6a of PS1 and substrate residues near the scissile bond(s) in the C83-bound (A) and Notch1 (N100)-bound (B) γ-secretase cryo-EM structures (PDB 6IYC and PDB 6IDF, …
Figure 6—figure supplement 2
Biophysical properties of DDM and POPC.
(A) Structures of DDM and POPC. (B) Calculated lipid order parameters SCH of the acyl chains of DDM (red) and POPC (sn-1 blue, sn-2 black). Data are represented as mean ± SD (n=3 trajectories). (C) …
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Figure 6—figure supplement 2—source data 1
Raw values of simulation data analysis (Source data for Figure 6—figure supplement 2B, C).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig6-figsupp2-data1-v2.zip
Figure 6—figure supplement 3
MD simulations in different DDM micelles.
(A) PSH with bound C83 substrate embedded in a DDM micelle environment (225 DDM molecules). (B) Calculated lipid order parameters SCH of the acyl chains of DDM in small (red, 150 molecules) and …
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Figure 6—figure supplement 3—source data 1
Raw values of simulation data analysis (Source data for Figure 6—figure supplement 3B-G).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig6-figsupp3-data1-v2.zip
Figure 7 with 1 supplement
Impact of PSH TMD6a mutations on PSH structural dynamics and activity.
(A) RMSD of the WT (blue) and the mutated systems M172K (violet), I173K (green), L175K (gray), and A176K (orange) in the POPC bilayer environment. The solid, dashed, and dotted lines represent three …
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Figure 7—source data 1
Raw values of simulation data analysis (Source data for Figure 7A–E).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig7-data1-v2.zip
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Figure 7—source data 2
Immunoblot images (raw and annotated) of cleavage assays (Source data for Figure 7F).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig7-data2-v2.zip
Figure 7—figure supplement 1
Secondary structure of lysine-mutant PSH TMD6a and surrounding residues over simulation time.
Secondary structure of PSH TMD6a (orange) and surrounding residues bearing lysine mutants in POPC bilayer environments over simulation time calculated using DSSP.
Figure 8 with 3 supplements
Stabilization of the PSH active site geometry in a POPC bilayer.
(A) Histograms of the Cγ-Cγ distances between the D162 and D220 of PSH measured in DDM micelle (red) and POPC bilayer (blue) environments. The dashed line indicates the distance of 7 Å. The measured …
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Figure 8—source data 1
Raw values of simulation data analysis (Source data for Figure 8A and Figure 8—figure supplement 1A-C).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig8-data1-v2.xlsx
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Figure 8—source data 2
Immunoblot images (raw and annotated) of inhibitor precipitation assay and cleavage assays (Source data for Figure 8C, E-G).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig8-data2-v2.zip
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Figure 8—source data 3
Raw values of immunoblot quantitation (Source data for Figure 8D).
- https://cdn.elifesciences.org/articles/76090/elife-76090-fig8-data3-v2.xlsx
Figure 8—figure supplement 1
Cγ-Cγ distances in PSH homology model.
(A) The average Cγ-Cγ distances between D162 and D220 of PSH. Each data point stands for the average value throughout one trajectory with the error bar showing the SD of the three independent …
Figure 8—figure supplement 2
Detailed geometry of the E-S catalytic site.
Detail geometries and distances for the atoms between D162, D220 and C83 L49 at a Cγ-Cγ distance of 8.2 Å in DDM micelles (A) and 6.8 Å in POPC bilayer (B).
Figure 8—figure supplement 3
Chemical structures of GSIs.
(A, B) Structures of TSA GSIs L-685,458 (A) and III-31C (B). (C–F) Structures of non-TSA GSIs DAPT (C), LY411575 (D), Begacestat (E) and MRK-560 (F).
Appendix 1—figure 1
Quality control of WT and mutant PSH.
(A) Analysis of protein aggregation of WT and mutant PSH by DLS. (B) Analysis of protein aggregation of WT and mutant PSH by BN-PAGE followed by immunoblotting for PSH (6F4). (C) Analysis of protein …
Author response image 1
Comparison of PSH activity in the POPC bilayer with and without the addition of DDM.
Analysis of PSH activity in POPC vesicles with and without the addition of 0.008% DDM after incubation with C100-His6 at 37 °C overnight by immunoblotting for AICD (Y188) and Aβ (2D8). The asterisks …
Author response image 2
Cleavage of C99 by PSH reconstituted in brain lipid vesicles. (A) Analysis of PSH activity in DDM micelles, POPC vesicles and brain lipid vesicles after incubation with C100-His6 substrate at 37 °C …
Author response image 3
Binding of POPC and DDM molecules on the PSH surface.
(A) The residue-wise averaged solvent residence time of POPC (blue) or DDM (red) on PSH surface. (B) Visualization of the DDM (middle) and POPC (right) residence time based on (A) from two different …
Videos
Video 1
Detergent-enzyme interaction in the DDM environment.
600 ns trajectories of PSH (red) in complex with C83 (orange) and nearby DDM molecules. A DDM molecule enters into the gap between TMD2 and TMD6. TMD6a switches between a helical and a loop …
Video 2
Detergent-enzyme interaction in the DDM environment.
600 ns trajectories of PSH (red) in complex with C83 (orange) and nearby DDM molecules. A DDM molecule enters into the gap between TMD3 and TMD4. TMD6a switches between a helical and a loop …
Video 3
Lipid-enzyme interaction in the POPC environment.
600 ns trajectory of PSH (blue) in complex with C83 (orange) and nearby POPC molecules. POPC molecules do not enter between TMD gaps and TMD6a remains a stable helix throughout the whole trajectory.
Tables
Table 1
Templates used for model building of PSH in complex with C83.
Residues of PSH used for model building are indicated.
| Template | |||
|---|---|---|---|
| 6IYC(PS1, C83) | 4HYG(Chain B) | Model 1 | |
| Model 1 | * | – | – |
| Model 2 | – | L7-D162, D220-L292 | complete |
| Model 3 | – | L7-A176, E210-A293 | complete |
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*
PSH residues L7-R193 and E210-A293 were modeled based on the template.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21(DE3)RIL | Agilent Technologies | Cat# 230245 | |
| Recombinant DNA reagent | pQE60-C100-His6 | Edbauer et al., 2003 | N/A | |
| Recombinant DNA reagent | pQE60-C83-His6 | This study | N/A | |
| Recombinant DNA reagent | pET21b-PSH | Li et al., 2012 | N/A | Gift from Yigong Shi |
| Antibody | Anti-APP (C-terminus) Y188 (rabbit monoclonal) | Abcam | Cat# ab32136 | IB (immunoblot) (1:5000) |
| Antibody | Anti-APP (C-terminus) 6687 (rabbit polyclonal) | Steiner et al., 2000 | N/A | IP (immunoprecipitation) (1:100–1:200) |
| Antibody | Anti-APP (Aβ22–35) Aβ (22-35) (rabbit polyclonal) | Sigma-Aldrich | Cat# A3356 | IB (1:1000) |
| Antibody | Anti-APP (Aβ1–16) 2D8 (mouse monoclonal) | Shirotani et al., 2007 | N/A | IB (3 µg/ml) |
| Antibody | Anti-APP (Aβ17–24) 4G8 (mouse monoclonal) | BioLegend | Cat# 800702 | IB (1:500-1:2500) |
| Antibody | Anti-PSH (residues 192–204) 6F4 (rat monoclonal) | This study | N/A | IB (3 µg/ml), generation of antibody described further below |
| Antibody | Anti-Penta-His (mouse monoclonal) | Qiagen | Cat# 34660 | IB (1:1000) |
| Chemical compound, drug | Ni-NTA Agarose | Qiagen | Cat# 30210 | |
| Chemical compound, drug | Calbiosorb Adsorbent beads | Calbiochem | Cat# 206550 | Discontinued |
| Chemical compound, drug | POPC | Avanti Polar Lipids | Cat# 850457P | Powder |
| Chemical compound, drug | Rhodamine-DHPE | Invitrogen | Cat# L1392 | |
| Chemical compound, drug | Sephacryl S-200 HR | GE Healthcare | Cat# 17058410 | |
| Chemical compound, drug | Streptavidin Sepharose | GE Healthcare | Cat# 17511301 | |
| Chemical compound, drug | L-685,458 | Sigma-Aldrich | Cat# 565771 | InSolution γ-Secretase Inhibitor X, used in cleavage assays |
| Chemical compound, drug | L-685,458 | Sigma-Aldrich | Cat# L1790 | Powder, dissolved in DMSO and used in inhibitor affinity precipitation experiments |
| Chemical compound, drug | Merck C | Taros Chemicals | N/A | Biotinylated L-685,458 |
| Chemical compound, drug | n-Dodecyl β-D-maltoside (DDM) | Millipore | Cat# 324355 | |
| Chemical compound, drug | Protein G Sepharose | Cytiva | Cat# 17061801 | |
| Chemical compound, drug | Protein A Sepharose | Cytiva | Cat# 17528001 | |
| Chemical compound, drug | Tropix I-BLOCK | Invitrogen | Cat# T2015 | |
| Chemical compound, drug | III-31C | Sigma-Aldrich | Cat# C0619 | |
| Chemical compound, drug | DAPT | Boehringer Ingelheim Pharma KG | N/A | |
| Chemical compound, drug | LY411575 | Karlheinz Baumann | N/A | |
| Chemical compound, drug | Begacestat | Karlheinz Baumann | N/A | |
| Chemical compound, drug | MRK-560 | Karlheinz Baumann | N/A | |
| Commercial assay or kit | V-PLEX Plus Aβ Peptide Panel 1 (4G8) Kit | Meso Scale Discovery | Cat# K15199G | |
| Commercial assay or kit | NativePAGE 4 to 16%, Bis-Tris, 1.0 mm, Mini Protein Gels, 10 wells | Invitrogen | Cat# BN1002BOX | |
| Software, algorithm | GelAnalyzer 19.1 | Istvan Lazar Jr., PhD Istvan Lazar Sr., PhD, CSc | N/A | http://www.gelanalyzer.com |
| Software, algorithm | AMBER18 | Case et al., 2005 | N/A | |
| Software, algorithm | CHARMM-GUI | Jo et al., 2008 | N/A | |
| Software, algorithm | SWISS-MODEL | Waterhouse et al., 2018 | N/A | |
| Software, algorithm | PROPKA3.1 | Olsson et al., 2011; Sondergaard et al., 2011 | N/A | |
| Software, algorithm | DSSP | Kabsch and Sander, 1983; Touw et al., 2015 | N/A |
Table 2
pKa predictions as calculated by PROPKA3.1 for published PSH and γ-secretase structures.
pKa values of the catalytic aspartate residue which is most likely protonated are indicated in red.
| PDB ID | Enyzme | Ligand | pKa (D162, D220) |
|---|---|---|---|
| 4HYG | PSH | None | 5.04, |
| 4Y6K | PSH | III-31-C | 5.63, |
| PDB ID | Enyzme | Ligand | pKa (D257, D385) |
| 4UIS | PS1 | None | 3.18, |
| 5A63 | PS1 | None | 4.42, |
| 5FN5 | PS1 | None | , 3.63 |
| 5FN4 | PS1 | Unknown helix | 4.70, |
| 5FN3 | PS1 | Unknown helix | 4.90, |
| 5FN2 | PS1 | DAPT | 5.13, |
| 6IYC | PS1 | C83 | 6.39, X* |
| 6IDF | PS1 | Notch1 | 6.21, X* |
| 6LR4 | PS1 | Semagacestat | 6.12, |
| 6LQG | PS1 | Avagacestat | 6.08, |
| 7V9I | PS1 | L-685,458 | 7.11, |
| 7D8X | PS1 | L-685,458 and E2012 | 7.01, |
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*
For structure determination D385 was mutated to alanine and therefore no pKa value is given.
