Editor's evaluation: Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Cleavage of membrane proteins in the lipid bilayer by intramembrane proteases is crucial for health and disease. Although different lipid environments can potently modulate their activity, how this is linked to their structural dynamics is unclear. Here, we show that the carboxy-peptidase-like activity of the archaeal intramembrane protease PSH, a homolog of the Alzheimer’s disease-associated presenilin/γ-secretase is impaired in micelles and promoted in a lipid bilayer. Comparative molecular dynamics simulations revealed that important elements for substrate binding such as transmembrane domain 6a of PSH are more labile in micelles and stabilized in the lipid bilayer. Moreover, consistent with an enhanced interaction of PSH with a transition-state analog inhibitor, the bilayer promoted the formation of the enzyme’s catalytic active site geometry. Our data indicate that the lipid environment of an intramembrane protease plays a critical role in structural stabilization and active site arrangement of the enzyme-substrate complex thereby promoting intramembrane proteolysis. Editor's evaluation This work provides a strong contribution to our understanding of intramembrane proteolysis and in particular the subtle structural but significant influence of the lipid bilayer on proteolytic activity and coordination of the active site geometry. https://doi.org/10.7554/eLife.76090.sa0 Decision letter eLife's review process eLife digest Cutting proteins into pieces is a crucial process in the cell, allowing several important processes to take place, including cell differentiation (which allows cells to develop into specific types), cell death, protein quality control, or even where in the cell a protein will end up. However, the specialized proteins that carry out this task, known as proteases, can also be involved in the development of disease. For example, in the brain, a protease called γ-secretase cuts up the amyloid-β protein precursor, producing toxic forms of amyloid-β peptides that are widely believed to cause Alzheimer’s disease. Proteases like γ-secretase carry out their role in the membrane, the layer of fats (also known as lipids) that forms the outer boundary of the cell. The environment in this area of the cell can influence the activity of proteases, but it is poorly understood how this happens. One way to address this question would be to compare the activity of γ-secretase in the lipid environment of the membrane to its activity when it is entirely surrounded by different molecules, such as detergent molecules. Unfortunately, γ-secretase is not active when it is removed from its lipid environment by a detergent, making it difficult to perform this comparison. To overcome this issue, Feilen et al. chose to study PSH, a protease similar to γ-secretase that produces the same amyloid-β peptides but remains active in detergent. When Feilen et al. mixed PSH with lipid molecules like those found in the membrane and amyloid-β precursor protein, PSH produced amyloid-β peptides including those that are thought to cause Alzheimer’s. However, when a detergent was substituted for the lipid molecules this led to longer amyloid-β peptides than usual, indicating that PSH was not able to cut proteins as effectively. The change in environment appeared to reduce PSH’s ability to progressively trim small segments from the peptides. Computer modelling of the protease’s structure in lipids versus detergent supported the experimental findings: the model predicted that the areas of PSH important for recognizing and cutting other proteins would be more stable in the membrane compared to the detergent. These results indicate that the cell membrane plays a vital role in the stability of the active regions of proteases that are cleaving in this environment. In the future, this could help to better understand how changes to the lipid molecules in the membrane may contribute to the activity of γ-secretase and its role in Alzheimer’s disease. Introduction Intramembrane proteolysis is a crucial cellular mechanism underlying many fundamental physiological processes (Erez et al., 2009; Beard et al., 2019). It is also involved in pathological conditions, most prominently in Alzheimer´s disease (AD). Here, intramembrane cleavage within the transmembrane domain (TMD) of the amyloid precursor protein (APP) derived C99 substrate by γ-secretase results in the release of a variety of amyloid β-peptide (Aβ) species (Steiner et al., 2018). The longer forms, Aβ42 and Aβ43, are toxic to neurons and believed to trigger the onset of AD (Selkoe and Hardy, 2016). γ-Secretase is a membrane-embedded protein complex consisting of four components (Yang et al., 2017). The catalytic subunit presenilin is an aspartyl intramembrane protease (Wolfe et al., 1999; Li et al., 2000; Steiner et al., 2000; Steiner et al., 1999; Kimberly et al., 2000) present in the mammalian γ-secretase complexes as either presenilin-1 (PS1) or presenilin-2 variant (Yu et al., 1998; Saura et al., 1999). Mutations in PS1 are the major cause of familial AD (FAD) and cause an imbalance in the production of Aβ species that leads to relative increases of the longer forms over the normally major form Aβ40 (Steiner et al., 2018). Presenilins are evolutionary highly conserved proteins and related to the signal peptide peptidase (SPP) family of intramembrane proteases (Ponting et al., 2002; Weihofen et al., 2002). Ancestral precursors of presenilin and SPP exist in several archaea (Torres-Arancivia et al., 2010) and share key signature motifs including the protease family-defining GxGD active site motif (Steiner et al., 2000) with presenilin and SPP. The archaeal homolog from Methanoculleus marisnigri JR1 termed presenilin/SPP homolog (PSH) is capable of cleaving C99 and several other substrates (Torres-Arancivia et al., 2010; Dang et al., 2015; Naing et al., 2015; Naing et al., 2018). Similar to presenilin in the γ-secretase complex, PSH appears to cleave C99 in a sequential manner starting by initial ε-site cleavages between L49 and V50 (ε49) or T48 and L49 (ε48) followed by the release of various Aβ species from stepwise carboxy-terminal trimming cleavages (Dang et al., 2015; Takami et al., 2009). However, in contrast to presenilin, which requires complex formation with the other γ-secretase complex components for activity (Takasugi et al., 2003; Edbauer et al., 2003; Kimberly et al., 2003), PSH is active without accessory components. The crystal structure of PSH revealed first important insights into aspartyl intramembrane proteases showing that the two catalytic aspartate residues of the active site in TMD6 and TMD7 directly face each other and locate in a water-accessible cavity (Li et al., 2012). Subsequent cryo-electron microscopy (cryo-EM) structural analysis of γ-secretase showed that presenilin adopts a structure in the complex very similar to that of PSH (Sun et al., 2015) with nearly identical positions of the catalytic residues (Bai et al., 2015b). Further cryo-EM studies showed that binding of APP and Notch substrates causes major conformational changes in both enzyme and substrate (Zhou et al., 2019; Yang et al., 2019). These led to an enzyme-substrate complex (E-S) with an extended TMD6 by formation of a new and stable TMD6a helix as well as a hybrid β-sheet between enzyme and substrate that causes unfolding of the ε-cleavage site region in the substrate (Zhou et al., 2019; Yang et al., 2019). Interestingly, formation of the TMD6a helix was also observed by cryo-EM upon inhibitor binding thus partially mimicking the substrate-bound state (Bai et al., 2015a; Yang et al., 2021). The very similar structural folds of presenilin and PSH and the ability to cleave C99 in the TMD at the same sites as γ-secretase (Torres-Arancivia et al., 2010; Dang et al., 2015) make PSH an attractive model for the intrinsic protease activity of presenilin. To gain basic insights into the enzymatic workings of presenilin proteases, we thus set out to characterize the influence of two fundamentally different hydrophobic environments on the activity of PSH and asked if cleavage of C99 by the solubilized enzyme in detergent micelles would differ from a lipid-reconstituted state and if so, whether such differences could be correlated with the structural dynamics of this prototype presenilin protease or its E-S. Although the influence of lipids on the activity of presenilin and other intramembrane proteases is well documented (Paschkowsky et al., 2018), there are so far no studies in which biochemically determined activities of these proteases were linked with structural information that could explain how lipids, in particular a membrane bilayer environment, affect intramembrane protease structural dynamics and enzyme function. Since presenilins are not active in detergent micelles without lipids (Zhou et al., 2010), this critical question can however not be addressed for γ-secretase directly and requires a suitable model protease such as PSH. We found that detergent-solubilized PSH has a reduced carboxy-terminal trimming activity, that is processivity, compared to γ-secretase giving rise to an increased production of very long Aβ species such as Aβ46. Strikingly, the reconstitution of PSH into a lipid bilayer strongly promoted the protease processivity to shorter Aβ species such as Aβ38 highlighting the important role of the lipid membrane environment for intramembrane proteolysis. Furthermore, it enhanced the binding of a transition-state analog (TSA) γ-secretase inhibitor (GSI) affinity probe suggesting a more stable active site conformation in the lipid bilayer. These biochemical studies were accompanied by comparative modeling and molecular dynamics (MD) simulations to study the effect of detergent micelle and membrane lipid environment on substrate-bound PSH. In good agreement with the experimental data, the computational data suggest that the stabilization of TMD6a and the active site can explain the increased processivity and inhibitor binding in the membrane bilayer. Mutational analysis confirmed the assumed critical functional role of β-sheet and TMD6a corroborating the computational analysis of substrate-bound PSH. Collectively, these data provide insights into how structural adaptations occurring in response to changes in the hydrophobic environment from a micellar membrane mimetic to a real lipid bilayer translate into activity changes of an intramembrane protease. Moreover, with general implications for intramembrane proteolysis, they show how a lipid bilayer allows the formation of a stabilized active site geometry poised for substrate cleavage. Results PSH cleaves APP C99 to longer Aβ species To get insights into the intrinsic protease activity of presenilin, we set out to further characterize the intramembrane cleavage of C99 by PSH (Figure 1A). Consistent with previous findings (Dang et al., 2015), n-dodecyl β-D-maltoside (DDM)-solubilized, His-affinity-purified PSH could cleave the C99-based APP C100-His6 substrate (Edbauer et al., 2003) as demonstrated by the generation of the APP intracellular domain (AICD) and Aβ cleavage products (Figure 1B). Cleavage was inhibited by the TSA GSI L-685,458 (Shearman et al., 2000; Figure 1B) although much higher, micromolar concentrations were needed for efficient inhibition compared to those known for γ-secretase (Li et al., 2000; Shearman et al., 2000). Analysis of the Aβ profile using Tris-Bicine urea SDS-PAGE (Figure 1C) and MALDI-TOF mass spectrometry (Figure 1D, Figure 1—figure supplement 1) showed that Aβ40 and Aβ42 were the major Aβ species produced with a preference of Aβ42 over Aβ40. Interestingly, besides the increased generation of Aβ42 even longer Aβ species such as Aβ46 were relatively abundant. This suggests that PSH cleaves C99 at the same sites as γ-secretase but with reduced processivity. Figure 1 with 1 supplement see all Download asset Open asset 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 is indicated. (B) Analysis of PSH activity in DDM micelles after incubation with C100-His6 substrate overnight at 37 °C by immunoblotting for AICD (Y188) and Aβ (2D8). Specificity of substrate cleavage by PSH in the assay was controlled by sample incubation at 4 °C or 37 °C in presence of the GSI L-685,458 (20 µM). Immunoblotting of PSH (6F4) was performed to control for PSH levels. (C) Aliquot of samples from (B) separated by Tris-Bicine urea SDS-PAGE for identifying Aβ species produced by PSH in DDM micelles and analysis by immunoblotting (2D8). In (B) and (C), representative immunoblots from three to six independent biological replicates (i.e. independent protease preparations) are shown. (D) Representative MALDI-TOF MS spectrum of Aβ profile generated by PSH in DDM micelles from four independent biological replicates. The intensity of the highest peak was set to 100%. A GSI control is shown in Figure 1—figure supplement 1 and observed masses for identified Aβ species are shown in Figure 1—source data 1. 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 Download elife-76090-fig1-data1-v2.zip 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 Download elife-76090-fig1-data2-v2.xlsx Lipid membrane enhances the processivity of PSH We next investigated how a membrane environment of PSH influences the cleavage and processivity of the protease. Previous studies have shown that γ-secretase activity is dependent on the membrane environment as modulations of the lipid composition and/or bulk membrane properties in cell-free assays affected total activity as well as the ratios of the Aβ species generated (Osenkowski et al., 2008; Osawa et al., 2008; Holmes et al., 2012; Winkler et al., 2012). Furthermore, it was also shown that varying the pH in cell-free assays can modulate the total activity as well as the processivity of γ-secretase (Quintero-Monzon et al., 2011). To investigate whether and how PSH cleavage of C99 would respond to a change from the micellar environment in DDM to a lipid bilayer environment, PSH was reconstituted in defined small unilamellar vesicles (SUVs) composed of palmitoyl-oleoyl PC (POPC), the most abundant phospholipid of biological membranes (Figure 2—figure supplement 1A, B). We then performed PSH in vitro assays with DDM-solubilized or POPC-reconstituted PSH in a pH range from 5.5 to 9.0. As shown in Figure 2A, the total activity was highest in the mild acidic to mild alkaline pH range and sharply dropped at pH values above 8.0. The pH optima for both conditions were very similar and lying around pH 7.0. However, compared to the DDM-solubilized enzyme, the processivity of PSH was strongly promoted in the lipid bilayer environment of the POPC SUVs as seen by a strongly increased production of Aβ38 and Aβ40 and the strong reduction of longer Aβ species (Figure 2B). Interestingly, the processivity of the reconstituted PSH appeared to be more reduced at alkaline pH values as judged from the appearance of Aβ species longer than Aβ42 (Figure 2B) at pH 7.5 and higher. A direct comparison of the Aβ profiles at pH 7.0 confirmed the increased processivity of PSH in the POPC lipid bilayer (Figure 2C, Figure 2—figure supplement 2A). Finally, we investigated the initial ε-site cleavages of C99 by PSH in DDM micelles or POPC vesicles at this pH. Mass spectrometry analysis showed that C99 was cleaved in both conditions at the ε49 and ε48 cleavage sites resulting in the release of the two N-terminally distinct AICD50 (ε49) and AICD51 (ε48) species (Figure 2D, Figure 2—figure supplement 2B). Collectively, these data show that the lipid environment increases the processivity of PSH in cleaving C99. Figure 2 with 2 supplements see all Download asset Open asset 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 (6F4) was performed to control for PSH levels. (B) Separation of Aβ species produced by PSH in DDM micelles and POPC vesicles by Tris-Bicine urea SDS-PAGE and analysis by immunoblotting for Aβ (2D8). In (A) and (B), representative immunoblots from six independent biological replicates are shown. Confirmation of PSH reconstitution in POPC SUVs is shown in Figure 2—figure supplement 1. (C, D) MALDI-TOF MS analysis of Aβ (C) and AICD (D) species generated by PSH in DDM micelles and POPC vesicles at pH 7.0. Representative mass spectra from four independent biological replicates are shown. The intensity of the highest peak was set to 100%. GSI controls are shown in Figure 2—figure supplement 2 and observed masses for identified Aβ and AICD species are shown in Figure 2—source data 1. 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 Download elife-76090-fig2-data1-v2.zip 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 Download elife-76090-fig2-data2-v2.xlsx Enhanced processivity of PSH is independent of the APP substrate N-terminus Since the activities of DDM-solubilized and reconstituted PSH clearly differed, particularly in the processivity, we next sought to understand the underlying basis for this behavior at the level of its structural dynamics. Since there is no structure of PSH in complex with C99 available and structural investigations on γ-secretase in complex with an APP substrate were so far only performed with C83, an N-terminally shorter alternative C-terminal APP fragment generated by α-secretase (Lichtenthaler et al., 2011), we first tested whether C83 is processed similarly to C99. We thus analysed its cleavage by PSH in DDM micelles and the POPC bilayer at pH 7.0. C83 was cleaved by PSH in both conditions resulting in the generation of an AICD and the Aβ-equivalent cleavage product p3 (Lichtenthaler et al., 2011; Figure 3A). As judged from the processivity-reflecting ratios of p3 species ending at position 40 and 42, in contrast to the DDM micelle environment, the POPC bilayer enhanced the processivity and caused an increase in the relative production of shorter p3 species (Figure 3B). The increase in processivity was similar to that observed under these conditions for the corresponding Aβ species produced from C99 (Figure 3C). These data show that the increased processivity in the POPC environment is independent of the N-terminus of the substrate and that C83 and C99 behave comparable in both environments, so that C83 should be suitable as C99 surrogate for structural modeling and molecular dynamics simulations of PSH in complex with a substrate using information from the C83-bound γ-secretase. Figure 3 Download asset Open asset 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)). Immunoblotting of PSH (6F4) was performed to control for PSH levels. (B, C) p3-40/p3-42 ratio (B) and Aβ40/Aβ42 ratio (C) from PSH activity assays in DDM (red) and POPC (blue) environment analyzed by ECL-IA. Quantitative data are represented as mean ± standard deviation (SD) (n=3 biological replicates). Source data are shown in Figure 3—source data 1. 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 Download elife-76090-fig3-data1-v2.zip 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 Download elife-76090-fig3-data2-v2.xlsx Structural modeling shows key features of substrate-bound γ-secretase in PSH Since no experimental structure of the substrate-bound (holo) PSH is available, we generated 3 different starting structures, models 1, 2 and 3, for the PSH holo form using template-based modeling by assuming that PSH binds its substrate in a similar way as PS1. Since the PSH crystal structure (Li et al., 2012) (PDB 4HYG) misses several residues and loop segments in its substrate-free (apo) form, it is necessary to include the cryo-EM structure of holo PS1 bound to the C83 γ-secretase substrate (Zhou et al., 2019) (PDB 6IYC) as an additional template. In model 1, we used the entire holo PS1 including the C83 substrate as template whereas in models 2 and 3 most of the PSH structure was and only different of segments were on the template the C83 see Materials and methods for The of our holo PSH models are to the determined PSH crystal structure (Li et al., 2012) but include several residues and loop segments that are in the crystal structure 1, Figure supplement 1A, Figure supplement In as for model 2 (Figure it the TMD6a helix C-terminal of TMD6 and the hybrid β-sheet between the and the of the which is stabilized by with (Figure These two structural elements were also found in the γ-secretase complexes with bound C83 (Zhou et al., 2019) or (Yang et al., 2019) (PDB and were not present in the substrate-free γ-secretase In a between and in the PSH (Figure the and of the substrate-bound γ-secretase structure (Figure key features for substrate interaction and hybrid known from the complexes are found in our models of the Figure 4 with 2 supplements see all Download asset Open asset PSH (A) of the holo form of PSH (blue) with APP C83 substrate and the crystal structure of PSH (PDB 4HYG) in the form in and of all three models and their values are in Figure supplement and (B) Schematic of the between of the substrate and of PSH (C) of and TMD6a in the C83-bound PSH model residues and (D) of and TMD6a residues and in the C83-bound γ-secretase cryo-EM structure (PDB Analysis of and PSH activity in DDM and POPC environment after incubation with C100-His6 substrate at 37 °C overnight by immunoblotting for AICD (Y188) and Aβ (2D8). Immunoblotting of PSH (6F4) was performed to control for PSH levels. The two substrate which are independent of PSH cleavage. Figure data 1 Immunoblot images (raw and annotated) of cleavage assays (Source data for Figure Download 1 used for model of PSH in complex with of PSH used for model are indicated. PSH residues and were on the template. of the in the substrate-bound PSH models Comparative modeling is to the of the and how the are A template with quality or a with can to an protein it is necessary to the presence of specific structural features in our substrate-bound PSH models that were observed in the substrate-bound PS1 In of the of the impaired the activity of the enzyme C83 and (Zhou et al., 2019; Yang et al., 2019). To investigate whether this structural is of similar functional in PSH, we residues and within the PSH to Similar as for PS1 (Zhou et al., 2019; Yang et al., we also residues to and to When for their enzymatic activity, the three as well as the two showed and activities compared to PSH in both DDM micelle or POPC bilayer conditions (Figure These results suggest that as the residues to are important for the activity of the protease they form the observed in our structural Comparative molecular dynamics simulations of PSH in micelle and membrane environment reduced PSH in the lipid bilayer To get into the molecular on how micelle and membrane environments influence PSH conformational the C83-bound PSH models were in DDM micelle DDM or POPC bilayer POPC environments and in each three simulations with different of starting were performed In six were and were generated in of the PSH holo form in both environments are shown for model 2 in Figure to the starting with relative to the structure of or than and an for all model 2 simulations (Figure supplement 1A). were observed for all model 1 simulations and for for model It is that model 1 is than models 2 and 3 it is entirely on the PS1 template structure and is structural information from PSH. Figure with 3 supplements see all Download asset Open asset simulations of holo PSH forms in DDM micelle or POPC bilayer. (A) PSH with bound C83 substrate in a DDM micelle environment and a POPC bilayer (B) The of between the of C83 and the of PSH. data for the and the the of the mean of three data (C) The of C83 of different models in DDM (red) and POPC (blue) environments over three The areas the of the (D) the substrate TMD residues from the simulations of the holo PSH in DDM (red) and POPC (blue) environments for a is as the mean of molecules within of of the The indicate the position of the two ε-cleavage The the of the mean (n=3 The of PSH of different models in DDM (red) and POPC environments over three that residues are not in our PSH The TMD6a and the areas the of the on of residues to are shown in Figure supplement Figure data 1 Raw values of data analysis (Source data for Figure Download We first analyzed the substrate and interaction with PSH the simulations in both substrate binding the active site region the interaction of the with the at the of the C83 which is for substrate cleavage. The β-sheet interaction in of was found to be similar in both the micelles and membrane environments for model 3 where the are more in the

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