Journal of Cell Science Accepted...
Transcript of Journal of Cell Science Accepted...
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Title:
The dynamic conformational landscape of γ-secretase
Authors:
Nadav Elad1,2
, Bart De Strooper1,2, 5,#
, Sam Lismont1,2
, Wim Hagen3, Sarah Veugelen
1,2 Muriel
Arimon4, Katrien Horré
1,2, Oksana Berezovska
4, Carsten Sachse
3,# and Lucía Chávez-
Gutiérrez1,2,#
1 VIB Center for the Biology of Disease, 3000 Leuven, Belgium
2 Center of Human Genetics, University Hospitals Leuven & Department of Human Genetics,
KU Leuven, and Leuven Research Institute for Neuroscience and Disease (LIND), 3000 Leuven,
Belgium
3 European Molecular Biology Laboratory, Structural and Computational Biology Unit,
Meyerhofstr.1, 69117 Heidelberg, Germany
4 Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston,
MA, USA
5 UCL Institute of Neurology, Queen Square, London, UK
# Corresponding authors:
Bart De Strooper, MD, PhD: VIB center for the biology of disease, Herestraat 32 3000 Leuven,
Belgium. +3216373246 [email protected]
Carsten Sachse, PhD: [email protected]
Lucía Chávez-Gutiérrez, PhD: [email protected]
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t© 2014. Published by The Company of Biologists Ltd.
JCS Advance Online Article. Posted on 12 December 2014
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Abstract
The structure and function of the γ-secretase proteases are of vast interest because of their critical
roles in cellular and disease processes. We established a novel purification protocol for γ-
secretase complex that involves a conformation and complex-specific nanobody, yielding highly
pure and active enzyme. Using single particle electron microscopy, we analyzed the γ-secretase
structure and its conformational variability. Under steady state conditions the complex adopts
three major conformations, which are different in overall compactness and relative position of the
nicastrin ectodomain. Occupancy of the active or substrate binding sites by inhibitors
differentially stabilize sub-populations of particles with compact conformations, whereas a
Familial Alzheimer Disease-linked mutation results in enrichment of extended-conformation
complexes with increased flexibility. Our study presents the γ-secretase complex as a dynamic
population of inter-converting conformations, involving rearrangements at the nanometer scale
and high level of structural interdependence between subunits. The fact that protease inhibition or
clinical mutations, which affect Aβ generation, enrich for particular subpopulations of
conformers indicates the functional relevance of the observed dynamic changes, which are likely
instrumental for highly allosteric behavior of the enzyme.
Keywords:
γ-secretase / Alzheimer disease / Conformational landscape / Regulated intramembrane
proteolysis / Single particle electron microscopy
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Introduction
γ-Secretase complexes have the remarkable property to cleave integral membrane proteins within
the lipid bilayer. Proteolytic products provide signals to the extra- and the intracellular
environment, coordinating physiological and pathological processes at both sides of the cell
membrane (De Strooper and Annaert, 2010; Selkoe and Wolfe, 2007). Best studied are the roles
of γ-secretases in the processing of the amyloid precursor protein (APP) and the secretion of the
neurotoxic amyloid β (Aβ) peptides in the context of Alzheimer disease (AD). However, other
substrates, including Notch, N-cadherin and ErbB4, link γ-secretase activities to development,
cancer and immunity (De Strooper and Annaert, 2010; Selkoe and Wolfe, 2007).
Presenilin 1 (PS1), nicastrin (NCT), presenilin enhancer 2 (PEN-2) and anterior pharynx
defective 1A (APH-1A) assemble in a tetrameric complex (Fig. 1A) (De Strooper, 2003) and PS1
autoproteolysis results in an active pentameric γ-secretase (Thinakaran et al., 1996), in which the
catalytic center is structured at the interface between the amino- and the carboxy-terminal
fragments of PS (NTF and CTF respectively) (Esler et al., 2000; Li et al., 2013; Li et al., 2000;
Wolfe et al., 1999) and connected to the intracellular aqueous environment (Sato et al., 2006;
Tolia et al., 2006). NCT, a type 1 integral membrane glycoprotein, plays a critical role in
complex maturation and stabilization (Chávez-Gutiérrez et al., 2008) and might be involved in
substrate binding (Shah et al., 2005; Zhang et al., 2012), although this is debated (Chávez-
Gutiérrez et al., 2008). The recent crystal structure of the NCT ectodomain (ECD) shows a two-
lobed architecture and an interface of van der Waals and hydrogen bonds between them (Xie et
al., 2014). APH-1 and PEN-2 contribute seven and two transmembrane domains (TMDs) to the
protease complex respectively and their roles are poorly understood. The first high resolution
(4.5Å) structure of γ-secretase was very recently obtained using single particle electron
microscopy (EM) (Lu et al., 2014). The model presents NCT ectodomain and traces the 19 TMDs
organized in a horseshoe shaped membrane core, the flexible loops connecting the TMDs and the
hydrophilic N- and C- termini of PS1 and PEN-2 were not visualized due to their high flexibility.
Although it represents a major step towards the molecular understanding of γ-secretase
mechanisms, additional functional-structural information on the complex is needed to understand
how γ-secretase cleaves different substrates and how Alzheimer causing mutations affect its
activity.
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γ-Secretase is a highly regulated allosteric enzyme with at least two conformations under
equilibrium: differential subunit composition (Serneels et al., 2009), aging-related
posttranslational modifications by oxidative stress (Guix et al., 2012; Wahlster et al., 2013),
pathogenic mutations in PS1 (Berezovska et al., 2005), and interaction with substrates or γ-
secretase modulators (GSMs) (Uemura et al., 2010), all shift γ-secretase conformational
equilibrium and regulate protease activity. With the exception of Li and colleagues (Li et al.,
2014), previous single-particle EM studies reported individual structures providing static views
of the complex (Lazarov et al., 2006; Li et al., 2014; Lu et al., 2014; Ogura et al., 2006;
Osenkowski et al., 2009; Renzi et al., 2011). Li and colleagues (Li et al., 2014) reported
differences in the relative position of the NCT ECD in the presence or absence of a non-active
site inhibitor (compound E), providing the first glimpse of structural changes triggered by a non-
active site inhibitor. However, the study focuses on the characterization of single conformations,
rather than on the distribution of (multiple) conformations under varying conditions (“allosteric
ensemble”).
Based on the highly allosteric behavior of γ-secretase, we postulate that this multimeric complex
adopts multiple conformations in steady state conditions and that ligand (inhibitor, substrate,
allosteric modulators) binding or presence of a pathological mutation (mutant PS) in its active
site alters the equilibrium between conformers. In this view, the catalytic activity of γ-secretase is
defined by the “average activity” of a set of co-existing conformers, which equilibrium depends
on presence of ligands, mutations, lipids, etc. To test our hypothesis we purified γ-secretase
using a complex-specific nanobody (Nb) and analyzed the resulting pure and active γ-secretase
by single particle EM. We show that wild-type γ-secretase complex exists as an ensemble of at
least three distinct major conformations (compact, intermediate and extended), which are in
dynamic equilibrium. Inhibition, substrate binding or the PS1 Familial Alzheimer Disease
(FAD)-causing mutation delta Exon 9 remodel the conformational landscape of the protease by
enriching specific subpopulations that already preexist in the ligand-free state.
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Results
A novel purification protocol based on an anti-γ-secretase complex-specific nanobody
results in highly pure and active enzyme
We expressed γ-secretase in insect cells using a baculovirus expression system. We used one step
purified γ-secretase complex (Fig. 1B, lane 1 and 1C, lane 1) (Acx et al., 2014) to generate
single chain camelid anti-γ-secretase nanobodies (Nbs) (Pardon et al., 2014). We identified the
conformational Nb30, which immunoprecipitates γ-secretase in 1% CHAPSO, a condition that
preserves integrity and activity of the complex, but fails to immunoprecipitate any of the γ-
secretase subunits in 1% Triton X-100 (TX-100) (complex dissociating conditions) (Capell et al.,
1998) (Fig. 1E, F). Nb30 does not affect γ-secretase activity in an in vitro activity assay (data not
shown). Therefore it is unlikely that binding of Nb30 changes γ-secretase conformation, although
we cannot discard this possibility. We then incorporated Nb30 in the purification of active γ-
secretase (see material and methods). The protocol yields highly pure and active untagged γ-
secretase complex (Fig. 1B, lane 2 and 1C, lane 2). We also screened for detergents that keep γ-
secretase active and stable. Gel filtration to exchange CHAPSO to Lauryl Maltose-Neopentyl
Glycol (LMNG) results in active (Fig. 1D) and stable γ-secretase complex. A typical EM
micrograph of negatively-stained particles (Fig. 2A) shows that this purification procedure results
in monodisperse particles of the expected size.
In order to verify the integrity of the complex, we performed antibody labeling with antibodies
against NCT ECD, NCT C-terminus (NCT CT), PS1 CTF, APH-1 and PEN-2, imaged the bound
complexes by EM and performed 2D analysis. Class averages of antibody-bound enzyme and
free γ-secretase were aligned, enabling identification of the bound antibody as an extra density
protruding from the γ-secretase projection (Fig. 2B-D, more examples in supplementary material
Fig. S1). By analyzing the 2D class averages, we identify the extended density as the NCT ECD
(Fig. 2C and D, indicated in dark green). As expected, NCT CT antibodies (light green) were
found at opposing position to the NCT ECD antibodies. In the membrane core, APH-1 CT
domain antibodies were localized directly beneath the NCT ECD antibodies and the C-terminal
part of PS1-CTF antibodies close to the APH-1 epitope, but distant from the NCT ECD
antibodies. PEN-2 antibodies localize at a distant position from the extended domain. Notably, all
antibodies bound at 1:1 stoichiometric ratio, confirming the integrity of the complex.
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Wt γ-secretase complex exists as an ensemble of different conformers
We investigated the architecture of the γ-secretase complex by single particle EM. Data sets are a
priori low-pass filtered to 25 Å in order to avoid any bias of the alignment from noise. EM
images were analyzed in 2D using reference-free alignment and classification. The class averages
show variability in the membrane core and in the relative position of the extended domain density
(Fig. 3A). Since variability in 2D projections can result from different conformations or different
orientations, multiple initial 3D maps were reconstructed using the random conical tilt (RCT)
method (Radermacher et al., 1987) resulting in 3D structures with resolutions of 23 to 25 Å (Fig.
3B-D, supplementary material Fig. S2A-F). From a total of 30 3D classes we found three
different structural states: extended, intermediate and compact which account for 17%, 30% and
17% of the aligned structures respectively (Fig. 3E-G). Classes differ in overall compactness of
the complex, mainly seen as a large hinge rotation of NCT ECD towards the membrane core. We
obtained the same three significant structures using unsupervised 3D classification with RELION
software (Scheres, 2012) (supplementary material Fig. S2G), confirming both the overall
architecture of γ-secretase structures and the proposed conformational heterogeneity in the
preparation. The compact structure resembles previously published EM structures (Li et al., 2014;
Lu et al., 2014), including a membrane core, surrounded by a detergent belt and the NCT ECD.
Differences were found in the detergent belt, which appeared rougher and thicker in our
structures. This can be attributed to the different detergents used in addition to the negative stain
method. However, we noted that our obtained structures vary considerably in dimensions and
domain architecture, indicating that the complex fluctuates between different conformations
under steady state conditions.
We defined the length of the major axis as a good and simple indication of the population
diversity (Fig. 3H). Indeed, a dramatic 30% (5 nm) difference between the compact and extended
structures is observed along this axis. Extended, intermediate and compact are 16-17 nm, 14-15
nm and 12-13 nm long, respectively. Importantly, we provide evidence below that, similarly,
large differences in the conformation of the complex are observed under physiological conditions
in the cell membrane (Fig. 5 and 6).
Inhibitor binding to the γ-secretase complex shifts its conformational equilibrium
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We hypothesized that the equilibrium between the different conformations observed in our
experiments (defines protease activity at the macromolecular level) is shifted upon ligand binding
to the protease. We tested this by evaluating the shifts in the equilibrium between the different
conformers induced by binding of inhibitors to the protease. The transition-state analog inhibitor
X (inhX, L-685,458) and inhibitor peptide15 (pep15) bind the catalytic site and the initial
substrate site, respectively. Inhibitor binding, according to our hypothesis will stabilize particular
conformations and thus shift the conformational equilibrium. Indeed, class averages of the γ-
secretase-inhX complex (Fig. 4A, supplementary material S3B) appear rounded and less variable
at the NCT ECD density when compared to untreated γ-secretase (Fig. 3A). Accordingly, inhX
narrows the distribution and shifts the average length of the major axis from 15.3 nm (untreated,
Fig. 3H) to 12.8 nm (Fig. 4B, C, supplementary material S3A, B), indicating that inhibitor bound
γ-secretase is more compact and less structurally heterogeneous. The reduced structural
heterogeneity observed in the inhX-treated particles enabled us to validate the overall correctness
of the structure by tilt-pair analysis (Rosenthal and Henderson, 2003) (supplementary material
Fig. S4).
Next, we used a γ-secretase inhibitor pep15, which is a helical peptide that binds to the putative
initial substrate binding site in the complex (Das et al., 2003). Similar to inhX, pep15 enriches for
a compact conformation (Fig. 4D-F, supplementary material S3C, D), but NCT ECD is
positioned in the majority of the structures at the edge of the membrane core (Fig. 4E).
Accordingly, the average of the length of the major axis is 13.7 nm (Fig. 4F).
We asked whether the large conformational changes we observe in our in vitro system could also
be observed in γ-secretase complexes in their native state, when embedded in the cell membrane.
We therefore studied the effect of inhX on the conformation of the γ-secretase using
fluorescence-lifetime imaging microscopy (FLIM) on intact cells. We hypothesized that
treatment with inhX would increase the proximity of the NCT ECD to the membrane core and
therefore to its intracellular domain (Fig. 5A). Thus, we followed the interaction of antibodies
targeting NCT ECD and NCT CT (cytoplasmic domain) by FRET. Increased FRET efficiency in
HEK293 cells treated with 1 μM inhX, compared to non-treated cells, confirms that NCT ECD
approaches the membrane core (Fig. 5B).
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We wondered whether the increased compactness of the γ-secretase observed in the presence of
these two inhibitors of very different classes would affect the stability of the complex. We
therefore performed co-immunoprecipitation (coIP) experiments of endogenous γ-secretase,
using a nanobody against NCT ECD (Nb28) (Fig. 1E, F), in CHAPSO, which keeps the complex
together, or TX-100, which normally dissociates the complex (Fig. 6 lane 4). In agreement with
Barthet et al. (2011) (Barthet et al., 2011) we find that γ-secretase is stable in the presence of
inhX and partially stable in pep15 in TX-100 (Fig. 6 lanes 5 and 6). Thus, remodeling the
conformational landscape of γ-secretase upon inhibitor binding is observed in EM and translates
into γ-secretase stabilization in TX-100.
FAD mutation remodels the γ-secretase conformational landscape by enriching an extended
conformation
We wondered what the effect of a FAD mutation would be on the conformational landscape of
the complex. FAD mutation PS1 delta exon9 (Δ9) misses part of the extracellular loop between
PS1-CTF and -NTF and is therefore expected to provide visible alterations to the complex. It
should be noted that, similar to other FAD mutations, Δ9 results in an altered Aβ production
profile (Chávez-Gutiérrez et al., 2012; Perez-Tur et al., 1995). We expressed and purified FAD
Δ9 γ-secretase complex, collected and processed EM images as indicated above. Windowed
particles and class averages show the same features as wt-γ-secretase, indicating that the Δ9
complex maintains grossly the features of wild type γ-secretase (Fig. 7A). NCT ECD was visible
as a separate density in more than a third of the class averages and appeared remarkably mobile,
adopting different shapes and variable positions with respect to the membrane core (Fig. 7A and
B). This made 3D reconstruction very difficult and therefore, we limited our investigation by
analyzing the conformational variability in the γ-secretase-Δ9 using 2D eigenimage analysis (Fig.
7B-D) (Elad et al., 2008; Elad et al., 2007). Using this approach the global variability in the data
set is reduced by classifying the particles first based on their overall shape, allowing the
identification of more subtle local variability within classes as density peaks in eigenimages.
Interestingly, the occurrence of significant positive and negative density peaks indicates
systematic structure variation in the region of the NCT ECD, even in class averages that
contained no obvious density for the NCT ECD (due to high domain flexibility) (Fig. 7C, Δ9 last
image). Wt γ-secretase and inhX bound γ-secretase data sets show significantly weaker density
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peaks in eigenimage, although some variation in the NCT ECD region is observed in extended-
conformation classes of wt γ-secretase (Fig. 7C, yellow square). Finally, we confirmed an
enrichment of intermediate to extended conformations due to a highly mobile NCT ECD, as seen
in the length of the major axis (Fig. 7E, supplementary material S3E-G).
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Discussion
The “ensemble model” for allostery states that the diversity and distribution of conformations
present in solution underlies enzyme activity (Boehr et al., 2009; Motlagh et al., 2014). In this
view, the conformational landscape, rather than specific ‘active’ or ‘inactive’ conformations,
provides the basis for the biochemical function of proteins. The relevance of this hypothesis has
been demonstrated in structural studies, for example of the regulation of G protein-coupled
receptors (GPCRs) (Nygaard et al., 2013), the ribosome (Fischer et al., 2010), the transcription
factor TFIID (Cianfrocco et al., 2013), or the integrins (Takagi et al., 2002).
We present here a first characterization of the γ-secretase structure dynamics. The most
remarkable conclusion form our work is that γ-secretase exists as an ensemble of conformers
(Fig. 8). The equilibrium between conformers is modulated by inhibitor binding to the active or
substrate binding sites providing functional relevance to this observation. It is also interesting that
a mutation causing Alzheimer Disease increases the conformational heterogeneity of the
complex. Therefore, we suggest that clinical mutations in Presenilin might cause an overall
destabilization of the structure leading to increased flexibility. Such destabilization could
speculatively result in destabilization of the enzyme-substrate state and promote product release,
providing an explanation for the higher release of longer Aβ peptides from the complex in the
presence of FAD mutations (Chávez-Gutiérrez et al., 2012).
Importantly, independent biochemical experiments show that inhX and pep15 binding, which
resulted in increased compactness of γ-secretase in the EM images, dramatically affected the
biophysical stability of the complex in a dissociating detergent (TX-100). In addition, our FLIM
data supports the inference that such large conformational shifts of NCT ECD happen in intact
cells. In agreement, NCT ECD is critical for the assembly, maturation and stability of γ-secretase
(Chávez-Gutiérrez et al., 2008). Furthermore, several publications using FLIM showed how γ-
secretase undergoes conformational changes in the intact cell membrane (Berezovska et al., 2005;
Li et al., 2014; Lleo et al., 2004; Shelton et al., 2009; Uemura et al., 2010). Thus, molecular
interactions affect γ-secretase function by changing the frequencies of subpopulations in the
conformational landscape. Allosteric behavior, substrate affinity and the catalytic mechanisms
are therefore determined by the ensemble-average. Thus, the observed conformational landscape
truly reflects the inherent dynamics of γ-secretase.
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The observed dynamics have particular implications for future structural and functional studies.
Structural homogeneity is determinant for protein crystallization and the generation of high
resolution data by EM. It should be noted that the recently published high resolution structure (Lu
et al., 2014) is based on a careful selection of about 5% to 10% of the particles from the purified
material. We suggest that such ”in silico” purification and the use of amphipol masked potential
structural heterogeneity enabling to build the high resolution model. The observed heterogeneity
might also explain why different γ-secretase structures published until now (Lazarov et al., 2006;
Li et al., 2014; Ogura et al., 2006; Osenkowski et al., 2009; Renzi et al., 2011) are rather
divergent. The current study suggests that pre-incubation with inhX may help to reduce
heterogeneity. In addition, other ligands, e.g. nanobodies that stabilize the protease complex in
particular conformations may also help studies aiming to elucidate the atomic structure of the
complex.
On the functional side, the conformational fluctuations observed in the present study indicate that
a more complete description of the γ-secretase catalysis has to take into account the
interconverting conformers that may exist at each step of the catalytic reaction. i.e. the
conformational fluctuations give rise to ensembles of Michaelis-Menten Enzyme-Substrate (ES),
Enzyme-Product (EP) complexes and their corresponding transition states, and these ensembles
will undergo equilibrium shifts depending on environment. In fact, the concept that multiple
enzyme conformers catalyze reactions in parallel is studied by “catalytic networks” (Benkovic et
al., 2008). Incorporation of dynamics in the studies of γ-secretase intramembrane proteolysis is
thus critical for understanding its function and elucidating its structure at high resolution.
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Materials and methods
Antibodies
The following antibodies were used for labeling: affinity purified rabbit polyclonal anti-APH-1a
(B80.3) and affinity purified monoclonal anti-NCT CT 9C3 have been described (Annaert et al.,
2001; Esselens et al., 2004). Affinity purified rabbit polyclonal against N-terminal amino acids
103-124 of NCT (anti-NCT ECD, ab24741) and affinity purified rabbit polyclonal against 13
amino acids at the C-terminus of PEN-2 (anti-PEN-2, ab62514) were purchased from Abcam.
Rabbit polyclonal against C-terminal amino acids 450-467 of PS-1 (anti-PS1 CT, S182) was
purchased from Sigma. The following antibodies were used for detection in WB: Rabbit anti
PEN-2 (B126.1) (Annaert et al., 2001; Esselens et al., 2004), Anti human PS1–loop (MAB5232),
purchased from Chemicon, mouse anti-NCT from BD Transduction Laboratories and rat anti-PS1
NTF (MAB1563) from Millipore, anti-FLAG M2 from Sigma.
Immunoprecipitation with nanobodies
Hi5-cells overexpressing the γ-secretase complex were lysed in either 1% CHAPSO or 1% Triton
X-100 for 1h on ice. Unsoluble material was removed by ultracentrifugation 45 min at 100 000g.
100μg solubilized material protein was incubated with the indicated nanobodies, which were
previously covalently coupled to NHS-beads (GE Healthcare) accordingly to manufacturer
instructions. 25 µl Nb beads (0.3 mg Nb/ml beads) were used per condition.
Immunoprecipitations were carried out in PBS buffer containing 1% CHAPSO or 1% Triton X-
100 in a total volume of 250μl, overnight at 4°C. Bound and unbound fractions were analyzed by
SDS-PAGE and western blot.
Overexpression and purification of wt and Δ9 γ-secretase
γ-Secretase complex was overexpressed in Hi5 insect cells from a single vector carrying the four
subunits. NCT was expressed as a GFP fusion. Purification followed three steps (Fig. 1B, C).
Expression and first step purification using an anti-GFP nanobody were performed as recently
published(Acx et al., 2014). A second immune-affinity step, performed with a conformational
nanobody His tagged (Nb30) that recognizes the γ-secretase complex and not the individual
subunits (Fig. 1E, F), enriched for mature and active protease complex. Nb30/γ-secretase
complex was purified by immobilized metal ion affinity chromatography (IMAC) and eluted with
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250 mM imidazole. Finally, gel filtration in a Superose6 column (GE-Healthcare) removed
unbound Nb and exchanged detergent to 0.1% LMNG. Purified γ-secretase complexes were
tested in an in vitro activity assay as previously described (Acx et al., 2014).
FLIM-FRET
HEK293 cells were seeded at 150,000 cells per well on glass coverslips in 12-well. After 24h
medium was refreshed and 1 μM of L-685,458 (inhX, Calbiochem) or DMSO was added. After
overnight incubation, cells were fixed in 4% paraformaldehyde for 10 min, blocked (2% BSA,
2% FBS and 1% gelatin in PBS supplemented with 5% serum) and incubated with primary
antibodies (anti-NCT ECD ab24741 and anti-NCT CT 9C3) overnight at 4°C. Cells were
subsequently incubated with fluorophore (Alexa488 or Alexa555)-conjugated secondary
antibodies for 1 h at room temperature, washed with TBS and mounted in Vectashield H-1000
(Vector Labs).
The relative proximity between fluorophore labeled NCT ECD and CT was assessed by the
previously validated Fluorescence Lifetime Imaging Microscopy (FLIM) analysis as described
(Berezovska et al., 2005; Uemura et al., 2010). Briefly, the baseline lifetime (t1) of the Alexa488
fluorophore (negative control, FRET absent) was measured in the absence of an acceptor
fluorophore. Upon excitation of the donor fluorophore in the presence of Alexa555 acceptor
fluorophore, the donor lifetime shortens (t2) due to FRET if the donor and acceptor are less than
5-10 nm apart. The degree of donor fluorophore lifetime shortening (FRET efficiency) correlates
with the change in proximity between the two fluorescently labeled domains of NCT molecule.
The percent FRET efficiency is calculated using the following equation: %EFRET=100*(t1-t2)/t1.
A mode-locked Chameleon Ti:Sapphire laser (Coherent Inc., Santa Clara, CA) sending a
femtosecond pulse every ~12.5 nsec was used to excite the donor fluorophore (two-photon
excitation at 800 nm wavelength). Donor fluorophore lifetimes were recorded using a high-speed
photomultiplier tube (MCP R3809; Hamamatsu, Bridgewater, NJ) and a fast time-correlated
single-photon counting acquisition board (SPC- 830; Becker & Hickl, Berlin, Germany).
SPCImage software (Becker&Hickl, Berlin, Germany) was used to determine the donor
fluorophore lifetimes by fitting the data to one (negative control) or two (acceptor present)
exponential decay curves.
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Sample Preparation for EM
Purified γ-secretase sample was diluted 1:10 to 0.035 mg/ml in buffer without detergent (25 mM
PIPES pH 7.4, 150 mM NaCl, 5% Glycerol) immediately prior to fixation on the EM grid. We
diluted the detergent in order to reduce background noise in EM micrographs. γ-Secretase was
stable and active at the final concentration of LMNG (0.01%), which is 10x the critical micelle
concentration (Chae et al., 2010). Carbon-coated 400 mesh grids were glow-discharged and then
incubated 1 min with a 3 μl drop of 0.1 mg/ml inert nanobody, which has no affinity to γ-
secretase and no effect on the structural analysis due to its small size (15 kDa). We found that this
pretreatment of the carbon surface improved staining quality. 3 μl of purified sample was then
applied and negatively stained with 2% uranyl acetate. For preparation of the γ-secretase-inhX
and γ-secretase-pep15 complexes, purified sample was diluted in buffer containing 10 μM of
inhX or 1 μM pep15 respectively, and incubated for 1 h at room temperature. Antibody labeling
for EM imaging was performed during the second step of the purification while γ-secretase was
bound on the Nb30 beads. Beads with bound γ-secretase were incubated overnight at 4°C with
the specified antibodies followed by washing and elution with 250 mM imidazole.
EM Data Collection and 2D/3D image processing
Wt γ-secretase, γ-secretase-pep15 tilt pair (0° and 55°) and antibody-labeled γ-secretase data sets
were collected on a Gatan US4000 CCD camera at 47,000 magnification (1.8 Å/pixel) using a
Titan Krios (FEI, Eindhoven) operated at 200 kV and controlled using SerialEM (Mastronarde,
2005). γ-Secretase-inhX data set was collected on a TVIPS 4k x 4k CCD camera at 83,333
magnification (1.8 Å/pixel) using a Phillips CM120 Biotwin Ice operating at 120 kV. γ-Secretase
Δ9 data set and γ-secretase-inhX tilt pairs (0° and 25°, for tilt pair validation) were collected on
TVIPS F224HD 2k x 2k CCD camera (Gauting, Germany) at 113,744 magnification (2.1
Å/pixel) using a FEI T12 operating at 120 kV. Imaging was done using a defocus range of 0.7–
1.3 µm and an electron dose of 30 e-/Å
2. The contrast transfer function (CTF) was determined for
each micrograph using CTFFIND3 (Mindell and Grigorieff, 2003). Particles from micrograph of
all samples except antibody labeled data sets were selected interactively using EMAN2 (Tang et
al., 2007). Particles of the antibody labeled data sets were selected automatically using a template
matching algorithm implemented SPIDER (Frank et al., 1996; Rath and Frank, 2004). Particles
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were extracted to yield boxes of 256 x 256 pixel dimension, and phase corrected using SPIDER.
After CTF correction, the box size was cropped to 160 x 160 pixels, images were binned by a
factor of 2 and band-pass filtered between 25 Å and 200 Å. Filtered particles were subjected to
10 iterations of reference-free alignment and classification using IMAGIC (van Heel et al., 1996),
starting with classes of 20 images and gradually increasing the number of images per class to
about 500, depending on sample homogeneity.
For RCT reconstruction of the wt γ-secretase 9,774 tilt-pairs were selected interactively using
EMAN2. The 0° tilt particles were subjected to reference-free alignment and classification,
resulting in 30 classes. A total of 30 3D RCT maps were reconstructed from the corresponding
tilted particles and each 3D map was refined internally against its tilted particles using IMAGIC.
Initial RCT structures were then refined iteratively with decreasing angular increment against a
data set of 36,093 0° tilt particles using SPIDER. A cross-correlation threshold was applied every
round to select best matching images for each structure. Additionally, in order to minimize the
effect of overpopulated orientations on the angular distribution, the number of images per
orientation was limited for 3D reconstruction. In total 6000-8000 molecular views (~20% of the
data set) were included in each structure. The FSCs from ‘gold standard’ refinement half-sets
(Scheres and Chen, 2012) indicate resolutions from 23 to 25 Å at FSC=0.143 for wt structures
(supplementary material Fig. S2A, C, E). Structures that refined until convergence, showed the
common features as described in the results above, and displayed good match between class
averages and re-projections were subsequently aligned in 3D and subjected to 3D classification.
Most of the structures featured preferred orientation with the γ-secretase extracellular side
contacting the carbon support. Therefore, structures could be roughly aligned by manual rotation
about the axis perpendicular to membrane plane. Manual alignment was followed by 4 iterations
of automated 3D alignment using UCSF Chimera (Pettersen et al., 2004), in which a 3D global
average was calculated and individual structures aligned to the global average structure.
Structures showing poor cross-correlation were discarded during the process. Aligned 3D
structures were subjected to multivariate statistical analysis (MSA)-based hierarchical ascendant
classification using IMAGIC. Unsupervised 3D classification in RELION (Scheres, 2012) was
performed using the 3D global average, filtered to 80 Å, as an initial model. The low-pass filtered
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data was classified into 10 3D classes, which showed the same architecture and heterogeneity as
above, therefore independently confirming the results.
γ-secretase-inhX and γ-secretase-Δ9 data sets contained 9,267 and 11,443 particles respectively.
The 30 initial RCT models from the wt data (filtered to 50 Å) were used as starting models for
3D reconstruction of both data sets. We used the same refinement procedure as for the wt, except
that about 3000-4000 images were included in each structure. 22 and 25 of the γ-secretase-inhX
γ-secretase-Δ9 structures respectively were aligned and classified as described above for the wt γ-
secretase structures. Structures of all data sets were aligned to the same coordinates.
γ-Secretase-pep15 data set was collected and processed as described for wt γ-secretase above.
6777 tilt pairs were selected interactively and classified into 15 classes using reference-free
alignment, which were then used to reconstruct 15 3D maps using the RCT method. 13 maps
were used for 3D alignment and classification.
Antibody labeled data sets contained 10,000 particles each. Data sets were processed separately
by reference-free alignment and classification as described above, followed by classification into
classes of 20 images. Class averages of poor quality were discarded, and the remaining class
averages were aligned to class averages of the wt γ-secretase (non-labeled) data set using
SPIDER. We then selected class averages from the labeled data set showing the same γ-secretase
features as in the matching class average of the non-labeled data set, and containing an extra
density at the periphery. Only extra density appearing consistently at the same position relative to
γ-secretase density was interpreted as bound antibody.
Tilt pair test (Rosenthal and Henderson, 2003) for validation of the overall correctness of the
inhX-treated structure was performed on 46 particles taken from a single micrograph using
EMAN2 e2tiltvalidate procedure.
Fitting of the high resolution map as a rigid body (Lu et al., 2014) was done automatically using
UCSF Chimera (Pettersen et al., 2004), following by small manual adjustments of the NCT ECD
atomic coordinates position.
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Acknowledgements
This work was supported by the Fund for Scientific Research Flanders (FWO); research fund KU
Leuven; the Hercules Foundation, Federal Office for Scientific Affairs (IAP P7/16); a
Methusalem grant of the Flemish Government, VIB, IWT, the European Research Council (AdG
to BDS), BDS is the Arthur Bax and Anna Vanluffelen chair for Alzheimer disease. NE was
supported by Marie Curie and European Molecular Biology Organization (EMBO) fellowships,
and by the Foundation for Alzheimer Research (SAO-FRA). OB was supported by NIH
AG15379 grant. We also acknowledge an anonymous foundation.
Figure legends
Figure 1 - γ-Secretase purification using complex-specific Nb30.
(A) Membrane topology of the five γ-secretase subunits. Subunits include Presenilin 1 amino-
terminal (PS1-NTF) and carboxy-terminal fragment (PS1-CTF), which contain the catalytic
aspartates. Nicastrin (NCT), is a type 1 integral membrane glycoprotein of 130 kDa comprising a
large ectodomain (ECD), Anterior pharynx defective 1 (APH-1), is a 30 kDa protein of seven
TMDs topology, and presenilin enhancer 2 (PEN-2) is a 12 kDa hairpin-like protein with two
TMDs.
(B,C) Coomassie-stained SDS-PAGE and western blot following the three purification steps: (1)
Anti-GFP Nb affinity chromatography and elution by protease cleavage, (2) Binding to complex-
specific Nb30-His and IMAC, (3) Size-exclusion chromatography. Colored arrow heads mark the
different subunits according to the color scheme in panel (A).
(D) Activity assay of the purified γ-secretase as measured by AICD generation from APP
C99x3FLAG substrate. Increasing amounts of AICD product (lower lanes) is seen with
increasing concentration of purified γ-secretase (lanes 2-5). Additionally, purified γ-secretase is
inhibited specifically by transition-state analogue inhX (lane 1). E and F).
(E, F) Anti γ-secretase complex-specific Nb30. Nb30 immunoprecipitates (IPs) only the
assembled γ-secretase complex, whereas it fails to bind γ-secretase under dissociating conditions
(TX-100). Hi5 insect cells overexpressing γ-secretase complexes were solubilized in either 1%
CHAPSO (E) or 1% Triton X-100 (F) and used in IP experiments with Nbs 28, 30 and 125. 1%
CHAPSO keeps active γ-secretase complex, while 1% Triton X-100 dissociates the complex.
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Shown are SDS-PAGE/Western blot analyses of input, unbound and bound fractions.
Immunostainings for γ-secretase subunits indicate that Nb30 binds γ-secretase when the complex
is stable (1% CHAPSO, E), but not the individual subunits (1% Triton X-100, F). In contrast,
Nb28 binds both γ-secretase complex and dissociated NCT subunit. Nb125 is a non-interacting γ-
secretase Nb used as negative control.
Figure 2 - EM characterization of purified γ-secretase complex.
(A) Representative micrograph of the purified γ-secretase. Scale bar: 50 nm.
(B,C) Class averages of free (B) and antibody-labeled (C) γ-secretase showing corresponding
orientations. Extra density resulting from the bound antibody is pointed by an arrowhead for each
of the different antibodies in (C). The density of the anchored Fab is enhanced in class averages,
whereas other domains of the antibody, which move independently, are averaged out.
(D) Outlines of the densities in (B), γ-secretase is in grey and antibody density in colors.
Additional class averages in supplementary material Fig. S1.
Figure 3 - Variable conformation of wt γ-secretase.
(A) Representative class averages used for 3D reconstruction by the RCT method. Outlines of the
densities are shown next to class averages, membrane core in grey and NCT ECD in green.
Separation into domain is based on the reconstructed 3D maps.
(B-D) Representative structures from the different 3D classes - extended, intermediate and
compact. Structures are displayed from the side (viewing into the membrane plane). Cryo-EM
structure from (Lu et al., 2014) filtered to 30 Å is shown in the inset in grey for comparison.
(E-G) 3D class averages calculated by MSA-based hierarchical ascendant 3D classification. The
proportion of aligned RCT structures represented by each class is indicated.
(H) Length distribution of the γ-secretase structures based on measurements along their major
axis.
Figure 4 – Differential stabilization of the compact conformation induced by inhX and
pep15.
(A, D) Representative class averages from the γ-secretase-inhX and γ-secretase-pep15 data sets
respectively.
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(B, E) 3D averages and representative RCT structures from the γ-secretase-inhX and γ-secretase-
pep15 data set respectively, displayed from the side as the untreated γ-secretase structures (Fig
3B-G).
(C, F) Length distribution of the different γ-secretase-inhX nd γ-secretase-pep15 structures
respectively, based on measurement along their major axis.
Figure 5 - InhX-induced NCT ECD movement observed in HEK293 cells using FRET-
FLIM.
(A) The experimental setup is illustrated in the cartoon: FRET-FLIM was used to monitor the
distance between γ-secretase NCT ECD and NCT CT in HEK293 cells using antibodies carrying
donor and acceptor fluorophores.
(B) FRET efficiency was considerably higher following inhX treatment, indicating that the NCT
ECD has moved closer to the NCT CT on the cytosolic side. P = 0.0003 by unpaired Student’s t
test, bars represent std. error.
Figure 6 - InhX and pep15 treatments differentially increase the association of γ-secretase
subunits in HEK293 cells.
Co-IP of γ-secretase subunits from HEK293 cells using an anti-NCT nanobody (Nb28, Fig 1). IP
was either performed in Triton X-100 buffer following O/N incubation with 1 μM inhX (lanes 2
and 5) or 10 μM pep15 (lanes 3 and 6). InhX treatment stabilized the γ-secretase complex against
dissociation in TX-100, whereas pep15 induces partial stabilization compared to DMSO treated,
supporting the EM results.
Figure 7 - Conformational landscape altered by PS1-Δ9 mutation.
(A) Class averages comprising a small number of images (~10) per class, showing the expected
membrane core and NCT ECD domains as observed in the wt. The position of the NCT ECD
varies considerably.
(B) Illustration of the technique for identification of local variability in 2D classes: data-sets are
sorted into classes of similar features (represented in 2D class-averages). Then, in order to assess
local variability within classes, the images within each class are subjected to MSA and the
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resulting eigenimages examined. Strong positive and negative density peaks in eigenimages
indicate local variability.
(C) Representative class averages from the γ-secretase-Δ9 data set (left panel, first row) and
corresponding eigenimage analysis of the images within classes (left panel, second row). Green
squares mark the position of significant positive and negative density peaks in eigenimages,
indicating systematic local variation at these positions. The same analysis was done for the class
averages of the wt γ-secretase and γ-secretase-inhX (right panel), resulting in significantly
weaker density peaks in eigenimages, although some peaks were observed in the extended
conformation class averages (yellow square).
(D) Quantification of local variability in the wt γ-secretase (yellow), γ-secretase-inhX (blue) and
γ-secretase-Δ9 (green). Reference-free aligned images from the three data sets were classified
into 30 classes and eigenimages calculated within each class. Density of the highest and lowest
peaks were measured in eigenimages 2 and 3 (eigenimage 1 represents the total average). γ-
secretase-Δ9 shows significantly stronger density in peaks compared to wt γ-secretase and γ-
secretase-inhX, indicating local variations.
(E) Length distribution of the different γ-secretase-Δ9 structures (supplementary material Fig.
S3E-G) based on measurement along their major axis.
Figure 8 - Energy landscape view of γ-secretase
Schematic energy landscape view of the conformational diversity in wt γ-secretase and its
rearrangement in γ-secretase-inhX, γ-secretase-pep15 and γ-secretase-Δ9 populations. Wt γ-
secretase fluctuates between three major conformations. InhX and pep15 differentially stabilize
the most compact conformation observed in the untreated γ-secretase, whereas the Δ9 mutation
increases conformational flexibility and enriches the subpopulations with intermediate to
extended conformations. The overall conformational landscape is multidimensional, however
here it is reduced to one dimension, concentrating on the length of the major axis.
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l Sci
ence
Acc
epte
d m
anus
crip
t
C
B
D
A
Figure 2 Jo
urna
l of C
ell S
cien
ceA
ccep
ted
man
uscr
ipt
1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8
0
2
4
6
8
Length of major axis (nm)
H
# s
tru
ctu
res
Figure 3
D B
10
nm
2D class averages
3D class averages
C
Individual RCT structures
G E F
A
Extended 17%
Intermediate 30%
Compact 17%
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
A
Figure 4
B E
10
nm
# st
ruct
ure
s
Length of major axis (nm)
# st
ruct
ure
s
Length of major axis (nm)
γ-secretase + pep15 γ-secretase + inhX
D
F C
3D class average
RCT structure
3D class average
RCT structure
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
D M S O + In h X0
2
4
6
8
1 0
%E
FR
ET
***
Figure 5
A B
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
NCT
PS1 CTF
PS1 NTF
PEN-2
DM
SO
Inh
X
Pep
15
Input
IP: anti-NCT
Bound
DM
SO
Inh
X
Pep
15
Figure 6 Jo
urna
l of C
ell S
cien
ceA
ccep
ted
man
uscr
ipt
Figure 7
# st
ruct
ure
s
Length of major axis (nm)
Δ9
2D class- averages
Eigenimages
C
Eigenimage 2
Positive Negative
D
Den
sity
Positive Negative
Eigenimage 3
B Local variability
Eigen- image
2D class- average
NCT ECD Core
A 1
0 im
g/cl
ass
E
wt inhX
Jour
nal o
f Cel
l Sci
ence
Acc
epte
d m
anus
crip
t
Ener
gy
Conformations En
ergy
Ener
gy
Conformations
Ener
gy
pep15
Figure 8 Jo
urna
l of C
ell S
cien
ceA
ccep
ted
man
uscr
ipt