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Chemical crosslinking/mass spectrometry maps the amyloid # peptide binding region on both apolipoprotein E domains Stéphanie Deroo ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb500994j • Publication Date (Web): 29 Dec 2014 Downloaded from http://pubs.acs.org on January 14, 2015

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Chemical crosslinking/mass spectrometry maps the amyloid β peptide binding region on both apolipoprotein E domains

Journal: Manuscript ID: Manuscript Type: Date Submitted by the Author: Complete List of Authors:

ACS Chemical Biology cb-2014-00994j.R1 Article 10-Dec-2014 Deroo, Stéphanie; Université Libre de Bruxelles, Faculté des Sciences Stengel, Florian; ETH Zurich, Institut f. Molekulare Systems Mohammadi, Azadeh; Université Libre de Bruxelles, Faculté des Sciences Henry, Nicolas; Université Libre de Bruxelles, Faculté des Sciences Hubin, Ellen; University of Twente, Faculty of Science and Technology; Vrije Universiteit Brussel, Department of Biotechnology; VIB, Structural Biology Research Center Krammer, Eva-Maria; Université Libre de Bruxelles, Faculté des Sciences Aebersold, Ruedi; ETH Honggerberg, Institute of Biotechnology Raussens, Vincent; Université Libre de Bruxelles, Faculté des Sciences

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Chemical crosslinking/mass spectrometry maps the amyloid  peptide binding region on both apolipoprotein E domains

Stéphanie Deroo,†,¶ Florian Stengel,‡ Azadeh Mohammadi,† Nicolas Henry,† Ellen Hubin,║ Eva-Maria Krammer,† Ruedi Aebersold,‡,§ and Vincent Raussens†

†

Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles, Brussels, Belgium

‡

Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

§

Faculty of Science, University of Zurich, Zurich, Switzerland

║1

Nanobiophysics Group, MIRA Institute for Biomedical Technology and Technical Medicine, Faculty of

Science and Technology, University of Twente, Enschede, The Netherlands. 2Structural Biology Brussels, Department of Biotechnology, Vrije Universiteit Brussel, Brussels, Belgium. 3Structural Biology Research Center, VIB, Brussels, Belgium

¶

To whom correspondence should be addressed: E-mail: [email protected]



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ABSTRACT Apolipoprotein E (apoE) binds the amyloid  peptide (A), one of the major culprits in Alzheimer’s disease development. The formation of apoE:A complexes is implicated in both A clearance and fibrillization. However, the binding interface between apoE and A is poorly defined despite substantial previous research efforts and the exact role of apoE in the pathology of Alzheimer’s disease remains largely elusive. Here, we compared the three main isoforms of apoE (E2, E3 and E4) for their interaction with A1−42 in an early stage of aggregation and at near physiological conditions. Using electron microscopy and western blots, we showed that all three isoforms are able to prevent A fibrillization and form a non-covalent complex, with one molecule of A bound per apoE. Using chemical crosslinking coupled to mass spectrometry, we further examined the interface of interaction between apoE2/3/4 and A. Multiple high-confidence intermolecular apoE2/3/4:A crosslinks confirmed that Lys16 is comprised in the region of A binding to apoE2/3/4. Further, we demonstrated that both N- and C-terminal domains of apoE2/3/4 are interacting with A. The crosslinked sites were mapped onto and evaluated in light of a recent structure of apoE. Our results support binding of the hydrophobic A at the apoE domain-domain interaction interface which would explain how apoE is able to stabilize A and thereby prevent its subsequent aggregation.


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Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common cause of late-life dementia. Following the amyloid cascade hypothesis, AD has its origin in an imbalance between the accumulation and the clearance of a highly hydrophobic peptide, the amyloid  peptide (A) (1). A is a 38 to 43 residues long peptide which is produced by proteolytic cleavage of the amyloid precursor protein. A misfolding and aggregation are most probably the early events that ultimately lead to formation of amyloid fibrils and plaques, death of brain cells and AD symptoms such as memory loss and cognitive decline (2). The development of the most common, late-onset form of AD is influenced by genetic factors. Among them, the major risk factor is the APOE 4 allele corresponding to isoform E4 of apolipoprotein E (apoE) (3). Even though the precise implications of bearing this allele in AD pathogenesis are unclear, it has been proposed that distinct effects of apoE isoforms on A aggregation and/or clearance play a major role (4). ApoE is involved in lipid and cholesterol transport within the cardiovascular and central nervous system. In humans, apoE is a 299 amino acid long polymorphic protein with three major isoforms: E2, E3 and E4. They differ from one another by single amino acid substitutions. ApoE3, the most frequent form, contains a Cys and an Arg in positions 112 and 158, respectively, while apoE2 contains two Cys and apoE4 contains two Arg residues at these same positions. The presence of these Cys residues in apoE2/3 leads to the formation of disulfide-connected apoE molecules in cerebrospinal fluid, frontal cortex and hippocampus (5,6). Further, it has been shown that at micromolar concentrations, apoE exists in solution as a polydisperse mixture of monomers, dimers and tetramers (7). ApoE contains two independently folded structural domains connected via a flexible hinge region (residues 192−215) (8). The N-terminal domain (NTD) (residues 1−191) comprises the low density lipoprotein receptor and related receptors binding region, while the C-terminal domain (CTD) (residues 216−299) contains the major lipid binding region (9). In solution, the NTD is composed of an antiparallel 4-helix bundle and it includes the two mutation sites. It is suggested that Arg112 in apoE4 leads to distinct domain-domain interactions and



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reduced stability compared to apoE2/3 (10,11). Both properties have been related to the propensity of apoE4 to promote sporadic late-onset AD (12). Numerous mechanisms have been proposed to explain the increased AD risk of apoE4 carriers, among them several that involve A-dependent roles of apoE (4). Early studies demonstrated that apoE is present in cerebral amyloid deposits of AD patients (13), which in turn led to the proposal that apoE4 could promote A fibril growth more extensively than apoE2/3, acting as a pathological chaperone in amyloid deposit formation (14,15). Recently, the focus has shifted from amyloid fibrils towards A oligomers, as the current hypothesis states that soluble A oligomeric intermediates formed during the process of A aggregation represent the major toxic entities (2,16,17). Several lines of research now provide evidence for a link between apoE4 and A oligomers. Notably, it was recently demonstrated that apoE4 increases the stability of A oligomers in vitro (18), that the presence of apoE4 correlates with a higher A oligomeric content in brains from AD patients (19,20), and that apoE4 colocalizes with A oligomers at synapses (20). It is by now established that all apoE isoforms can interact with A in vitro (21). Yet, even if a direct protein-to-peptide interaction influencing the mechanism of A aggregation and/or deposition is generally accepted, the precise molecular nature of this interaction is challenging to define. A residues 1228 have been identified as necessary for apoE:A complex formation in vitro (22,23) and a recent study mapped A residues 17−22 to contain the main binding region for apoE:A interaction (24). However, the apoE region(s) involved in the interaction with A has not yet been clearly established. Most early studies, working with apoE and monomeric A, suggested that the binding site is located exclusively in the CTD of apoE (22,25,26) which cannot explain isoform-specific differences observed in apoE:A interaction (21), as the point mutations at positions 112 and 158 are situated in the NTD of apoE. However, both apoE N- and CTD were shown to mediate interaction with A fibrils and to do so with similar affinities (27). This study further showed that binding of the isolated NTD of apoE3 to A fibrils leads to a


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significant conformational change in this domain (27). Thus, the interaction site(s) between apoE and A is still not understood at a molecular detail. Defining and characterizing the precise interaction region(s) remains an important and open question in the field. All the more as it is known that other amyloidogenic peptides interact with the same undefined region of apoE (28). The aim of this study is to characterize in vitro the interaction of A with full-length apoE2/3/4 isoforms and to gain structural information on the binding region(s) involved. Specifically, working at a molar ratio of apoE to A close to physiological levels, we monitor the effects of the presence of apoE2/3/4 on A fibrillogenesis and evaluate apoE2/3/4:A complex formation. We subsequently investigate the apoE2/3/4:A binding site by chemical crosslinking coupled to mass spectrometry (CXMS). CX-MS is able to identify sites of interaction between different proteins and additionally provides low resolution distance constraints between crosslinked residues (29). In the case of the crosslinker used in this study, CX-MS will identify the Lys residues within and between apoE and A that are crosslinked upon incubation of apoE2/3/4 with A. We finally evaluate our results by mapping our high-confidence apoE:A crosslinks onto the structure of apoE (30) and discuss implications for the stabilization of hydrophobic A species in the context of apoE function.



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RESULTS AND DISCUSSION This work was performed using synthetic A1−42 (A), the highly amyloidogenic  peptide isoform, and recombinant human apoE. We were interested in defining whether there are differences in the interaction of A to the most abundant isoforms of apoE, namely E2, E3 and E4, and in investigating the non-covalent interface involved in complex formation between both partners. Most earlier in vitro studies performed with a high molar ratio of A to apoE, opposite to what is found in vivo (apoE:A molar ratio is 10−30 (31)), reported conflicting isoform-dependent complex formation characteristics (see (21) for a review). Therefore, we decided to study this interaction closer to physiological conditions using 2 molar equivalents of apoE2/3/4 to A under physiological pH and temperature (pH 7.4 and 37°C). ApoE2/3/4 interfere with A fibrillization. To assess the formation of A fibrils, we compared the morphology of A incubated alone or in the presence of 2 molar equivalents of apoE isoforms by negative staining transmission electron microscopy (TEM) (Figure 1). After 24h incubation at 37°C, A alone forms abundant unbranched fibrils that can be several micrometers long (Figure 1, panel A). The fibrils have a diameter of 8 to 10 nm which is in agreement with previously published data on A (32). Former studies, performed with large excess of A over apoE, showed formation of such fibrils in the presence of apoE (14,15). Contrary to these results, no fibrils are formed in the presence of apoE2/3/4 under the conditions used here and the considered timeframe (Figure 1, panel B). Instead, smaller particles are observed which range from spherical entities to thread-like structures and resemble native apoE isoforms incubated alone in the same conditions (Figure 1, panel C). Our findings indicate that these structures are likely due to self-associated apoE species rather than A aggregation products and that all apoE isoforms interfere with A fibril formation. Evaluation of apoE2/3/4:A complex formation. The inhibition of fibril formation, evidenced above by TEM, is suggested to be due to direct protein-to-peptide interaction between apoE and A (18,33). As we and others previously showed, the interaction between apoE and A monomers is SDS-stable and can be visualized by SDS-Page (18,22,23). We compared apoE2/3/4 for their ability to form an apoE:A


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complex when mixed at a 2:1 molar ratio with A and incubated for 24h at 37°C. We first analyzed the samples under reducing conditions and the gel was blotted and detected with an antibody against A (Figure 2 – a parallel gel was blotted and detected with an antibody against apoE, Supplementary Figure S1). The blot shows A monomers below 10 kDa, A trimers and tetramers around 15 kDa, as well as significant formation of a species at 40 kDa corresponding to an [(apoE)1(A)] complex (Figure 2). No significant differences in [(apoE)1(A)] complex formation capacity between apoE isoforms are revealed. Previously, the presence of reducing agent during SDS-Page was shown to disrupt the apoE:A complex (22). We thus repeated the previous experiments for all 3 isoforms under non-reducing conditions (Figure 3, panels A and B). Parallel western blots were incubated either with anti-apoE or antiA antibodies. The blot incubated with anti-apoE reveals monomers for apoE4 (Figure 3, panel A). Due to the presence of the Cys residue(s), mainly dimers are observed at 70 kDa for apoE3, while for apoE2 we observe up to five molecules of apoE that are disulfide-bonded (Figure 3, panel A). The blot incubated with anti-A antibody (Figure 3, panel B) reveals no change in regard to [(apoE)1(A)1] complex formation for the apoE4:A sample when reducing and non-reducing conditions are compared. For apoE3:A, in addition to the band containing the [(apoE)1(A)1] complex, two extra bands are observed around 70 kDa. We assign these bands to [(apoE)2(A)1 or 2]. Finally, for apoE2:A a discrete series of additional bands are observed under non-reducing conditions. We assign them to [(apoE)x(A)y] with x ranging from 1 to 5 and y ≤ x (Figure 3, panel B). A is thus able to bind apoE whether apoE is monomeric or multimeric mediated by disulfide bonding(s). A binds to both apoE N- and CTD. We then investigated the interaction of apoE isoforms with A under native conditions using a crosslinking reagent that is reactive towards primary amines (i.e., side chain of Lys residues and N-terminal amine in proteins). We first tested several ratios of crosslinker to apoE:A to optimize the formation of the [(apoE)1(A)] complex while keeping apoE-apoE intermolecular crosslinking to a minimum (Supplementary Figure S3). We then evaluated the outcome of



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the crosslinking reaction applied on apoE:A samples by SDS-Page using the optimized conditions and by comparing apoE isoforms E2, E3 and E4. Figure 4 shows the result of 4 molar equivalents of crosslinker applied to samples of apoE2/3/4:A in a 2:1 molar ratio after incubation at 37°C for 24h. The blot is detected with anti-Aa parallel gel was blotted and detected with anti-apoE antibody, Supplementary Figure S4). As previously demonstrated, some SDS-stable [(apoE)1(A)] complex is already observed at 40 kDa without adding the crosslinking reagent (Figure 4). Following crosslinking, the intensity of the band assigned to the [(apoE)1(A)] complex increases and higher molecular weight species are observed around 70 kDa that are assigned to crosslinked [(apoE)2(A)1

or 2]

complexes.

Overall, the crosslinking profiles under reducing conditions are similar for apoE2/3/4:A samples, suggesting again that A binding does not depend on apoE isoforms and the presence of disulfide bridge(s). To identify the region(s) of apoE isoforms that mediates interaction with A, we used isotopic CX-MS (34). Samples were incubated with the optimized concentration of isotopically labeled disuccinimidyl suberate (DSS) and subsequent in-solution tryptic proteolysis provided a mixture of crosslinked and noncrosslinked peptides. After an enrichment step for crosslinked peptides (35), these were subjected to LCMS/MS (35), and the data were subsequently analyzed by the xQuest/xProphet pipeline (36,37). As expected, the vast majority of identified high-confidence crosslinks are apoE-apoE interactions (Supplementary Table S1). Focusing on the crosslinks between apoE and A yielded 6 high-confidence intermolecular apoE:A crosslinks (Table 1, Supplementary Figure S5 and Table S2). Interestingly, out of the two Lys residues within A (Lys16 and Lys28), solely Lys16 is identified in apoE:A intermolecular crosslinks (Table 1 and Supplementary Table S2). Lys16 is situated in the broad apoE binding motif of A (residues 12−28) and lies next to the 17−22 region that has been mapped as the main binding element mediating interaction with apoE (22−24). Our intermolecular apoE:A crosslinks thus agree with the region of A that has been previously shown to be necessary for apoE binding.


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Among the 6 apoE:A crosslinks detected, 5 could be identified reproducibly for all 3 apoE isoforms (Table 1), indicating that the binding site between apoE isoforms and A monomer is most likely similar. It is suggested that A interacts with the CTD of apoE (22,25,26), though the precise location is unknown and recent data have proposed that A could interact at more than one site on the CTD of apoE (26). Although our apoE-apoE crosslinks indicate that all Lys residues in the apoE CTD are available to the crosslinking reagent (Supplementary Table S1), we could detect only one high-confidence apoE:A crosslink involving the CTD of apoE, on Lys242 (Table 1). This result agrees with a previous experiment that suggested that A interacts at the lipoprotein binding site of apoE (residues 220−266 (38)) and thereby compromises apoE lipid binding ability (26). Additionally, we consistently obtained at least 4 apoE:A crosslinks within the NTD of apoE which suggest an interaction between A and the NTD of apoE. Interestingly, these crosslinked lysines are situated on 3 different helices of the 4-helix bundle of apoE NTD (Figure 5, panel A). To validate our identified crosslinks and to better understand the three dimensional implications of apoE:A interaction as suggested by our CX-MS approach, we mapped the crosslinked apoE Lys residues onto a recent high resolution NMR structure of apoE (30) (Figure 5, panels B-D). In the NTD of apoE, the crosslinked Lys residues are localized on 2 different faces of the 4-helix bundle, delineated by helices 2−3 and helices 3−4, respectively (Figure 5, panel B). Further, 4 out of the 5 lysines that were identified in all apoE isoforms as being crosslinked to A (Lys95, 143, 157 and 242) are situated at the proposed domaindomain interaction interface in apoE, composed of NTD helices 3−4 and CTD helix 2 (Figure 5, panels C and D) (30). In this model it would therefore be possible for an A monomer to bind at this domaindomain interacting region of apoE and still have Lys16 of A accessible for crosslinking with these four apoE Lys residues. We also observed crosslinks between A and Lys72 and 75 of apoE (Table 1), both of which are not situated at the CTD/NTD interface in this recently proposed NMR structure (30). The CTD/NTD domain interaction reported by Chen and co-workers (30) actually differs from the suggested domain interaction



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localization proposed in an earlier model (39). In apoE4, point mutation C112R would induce a salt bridge between Arg61 and Glu255 and result in the proximity of apoE4 CTD and helix 2 of the NTD (10). It this case, A could be interacting at a domain-domain interface involving apoE CTD and another face of the NTD 4-helix bundle and the crosslinks between apoE Lys72/Lys75 and A would then be satisfied. While our identified high-confidence crosslinks cannot be reconciled with a single apoE:A interaction model, our results indirectly suggest that domain-domain interaction in apoE, which is still a matter of intense debate, could be of greater variability than anticipated. This high structural variability could be of importance for A binding capabilities of apoE. Discussion. The misfolding and aggregation of A is thought to be at the origin of AD. During the progression of A aggregation, soluble oligomeric intermediates are formed before and concomitantly to the deposition of insoluble fibrils and plaques. Following the amyloid cascade hypothesis, the current view pinpoints these soluble A oligomers as being responsible for synaptic dysfunction (2). Contradictory data tend to show that either apoE promotes A fibril formation (14,15), or rather stabilize these soluble forms of A resulting in a delay in fibril formation onset (18,19,40). The isoform-specific differences observed provide explanation of the higher AD risk linked to apoE4 (21). At an apoE to A molar ratio at near physiological conditions (2:1), we show here using TEM that apoE2/3/4 are all able to interfere with the aggregation process of A, preventing the formation of fibrils (Figure 1). Further, using SDS-Page in reducing conditions, we show by western blot that apoE isoforms, incubated with A in an early stage of oligomerization, are able to bind A monomers, leading exclusively to an SDS-resistant complex with a stoichiometry of [(apoE)1(A)] (Figure 2). These results support the recent view that apoE isoforms stabilize soluble forms of A, preventing the formation of fibrils. However at the low molar ratio of A to apoE used in this study, no isoform-specific effect could be demonstrated for any of the apoE isoforms. Although our results do not give evidence for a direct correlation between apoE isoforms and A stabilization/interaction in AD, additional factors need to be considered. Among them, it has been shown that apoE4 is a major risk factor for sporadic late-onset AD


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(3). Therefore an accumulation of A preceding the deleterious effects of apoE4 could be necessary and only when the A to apoE ratio reaches a certain threshold, apoE4 could start to stabilize oligomeric forms of A better than apoE2/3, as we previously demonstrated (18). Further, it is also crucial to understand apoE:A interaction in the context of apoE function. While in the blood circulation apoE is almost inexistent in a lipid-free fashion, the situation in the brain is less clear. It has been shown that apoE4 domain interaction was maintained in living neuronal cells and suggested it could decrease its lipid-binding capacity in comparison to apoE3 (41). This assumption was supported by recent studies indicating that levels of lipid-depleted apoE in the brain are higher for 4 allele carriers (42,43). Therefore, apoE4 may be more amenable than apoE2/3 to the type of direct protein-peptide interaction that was investigated in this work, which in turn could lead to more extensive stabilization of toxic A soluble species. Preventing apoE:A interaction has become a lead for the development of drug therapies against AD (44,45), but this requires the binding interface to be extensively characterized. Our CX-MS analysis enables us to delineate the interaction interface between apoE isoforms and A, which appears to be similar for all 3 isoforms in their lipid-depleted state. Though most previous in vitro studies using apoE and monomeric A located the A binding region solely within the apoE CTD (22,25,26), recent work showed that apoE interaction with A fibrils is mediated by both the N- and CTD of apoE (27). In situ measurements in AD brains further led to the proposal that apoE could interact with A in a similar way it interacts with lipids, leading to encapsulation of A in-between apoE N- and CTD (46). Our combined crosslinking data indicate that A Lys16 is in spatial proximity to apoE lysines located in both the apoE N- and CTD (Table 1 and Figure 5) when A is monomeric. We visualize our crosslinking data on a recent high-resolution model of monomeric apoE, which could be obtained be generating 5 mutations in the CTD of apoE (30). However these mutations do not seem to have any significant effect on the 3D conformation of apoE, as it was shown that mutant and wild type apoE share identical structure and stability, possess similar lipid-binding activities and identical receptor binding activity (47). The domain



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interaction in this mutant is also proposed to reflect the one encountered in the wild type (48). Mapping our high-confidence apoE:A crosslinks on this structure clearly supports an encapsulation mechanism as the majority of the intermolecular apoE:A crosslinks identified are situated at the domain-domain interface described (Figure 5). Our results thus suggest that monomeric A interacts at the apoE C- and NTD interface and could be pinched in between both the N- and CTD and therefore provide basis to explain the stabilization of A by apoE (Figure 1). This is highly relevant as it has been shown that apoE can form SDS-stable complexes with several other amyloidogenic peptides and that they compete for the same binding site (28). An identical type of sequestration of such hydrophobic peptides could therefore be envisioned as the one described here for A.


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METHODS Samples. We expressed and purified recombinant full-length E2, E3 and E4 isoforms of human apoE in Escherichia coli (ER2566 strain) following a previously described protocol (18). Synthetic A1−42 was purchased from American Peptide. Peptide films were prepared as previously described and stored at – 20°C (49). Homogeneous, unaggregated peptide solutions were obtained at a final A concentration of 1 mM after suspension of the peptide film in DMSO (Sigma) and careful vortexing. Non-aggregated A was then diluted in Hepes/NaCl buffer (20 mM Hepes, 150 mM NaCl, pH 7.4) and immediately supplemented with apoE isoforms in 20 mM Hepes, 150 mM NaCl, pH 7.4. Throughout the work we used mixtures of 6 M apoE and 3 M A (2:1 apoE:A molar ratio) which were incubated at 37°C for 24h. Controls of A or apoE isoforms incubated alone were always conducted in parallel. Transmission electron microscopy. A 10 L aliquot of each A, apoE:A and apoE sample (20 mM Hepes, 150 mM NaCl, pH 7.4) was spotted onto a carbon-coated Formvar 400-mesh copper grids (Agar Scientific) and incubated for 1 min. The grids were washed twice with water and stained with 1% (w/v) uranyl acetate. Samples were examined with a JEOL JEM-1400 transmission electron microscope at 80 kV. Images are representative of two independently prepared samples. Fibril diameters were measured manually using ImageJ (50). Electrophoresis gels and western blots. Samples in 20 mM Hepes, 150 mM NaCl, pH 7.4 were run on gradient 4−12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-Page) gels either in reducing or non-reducing conditions (Laemmli sample buffer, with or without -mercaptoethanol respectively) as stated in the main text. The protein bands were then transferred on nitrocellulose membranes for western blotting. Detection of A was carried out using 6E10 mouse monoclonal antibody (Covance) diluted 3000−5000 times in TBS-Tween20 with 0.1% (w/v) dry milk (performed on gels with 0.2 g A/lane). Detection of apoE was carried out using A1.4 mouse monoclonal antibody (Santa Cruz Biotechnology) diluted 3000−5000 times and performed on gels with 3 g apoE/lane. The anti-mouse secondary antibody horseradish peroxidase-conjugated (Millipore) allowed detection of protein bands



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using the Supersignal West Pico Chemiluminescent Substrate (Pierce). The associated luminescence was recorded using Image Quant TL 400 device (GE Healthcare). Controls of A and apoE2/3/4 incubated alone are shown in Supplementary Figures S1 and S2. Chemical

crosslinking.

To

visualize

the

effect

of

crosslinking

by

SDS-Page,

bis[sulfosuccinimidyl]suberate (BS3; ThermoScientific) was dissolved in water at a concentration of 5 mM. For CX-MS, an equimolar mixture of isotopically light (H12) and heavy (D12) labeled disuccinimidyl suberate (DSS; Creative Molecule Inc) was dissolved in DMSO at a concentration of 5 mM. The crosslinking reaction was performed by adding the appropriate amount of BS3 or DSS to preincubated apoE:A samples. For determination of crosslinking sites, 4 molar equivalents of DSS over apoE concentration were used and roughly 150 g of protein/peptide. The reaction mixture was incubated for 30 minutes at 37°C. Crosslinking was quenched by the addition of TRIS (Sigma) to a final concentration of 50 mM for 20 minutes at room temperature. In-solution digestion. 1.5 volumes of 50 mM ammonium bicarbonate were added for each volume of crosslinked proteins. Crosslinked proteins were reduced with 5 mM dithiothreitol (Sigma) at 95°C for 5 minutes and alkylated with 10 mM iodoacetamide (Sigma) at room temperature in the dark for 20 minutes. Proteins were digested with an enzyme-substrate ratio of 1 to 50 (w/w) with trypsin (Promega) at 37°C overnight. Mixtures were then evaporated until complete dryness in a vacuum centrifuge before further processing. Crosslinked peptide enrichment and mass spectrometry. Dry mixtures were re-suspended in 20 L of water/acetonitrile/trifluoroacetic acid (70:30:0.1 v/v/v) and crosslinked peptides were enriched using size exclusion chromatography (35), followed by LC-MS/MS analysis on a Thermo Orbitrap Elite mass spectrometer (Thermo Electron) (35). Data analysis was performed using xQuest (36) in iontag mode against a database containing apoE and A sequences with a precursor mass tolerance of 10 ppm. For matching of fragment ions, tolerances of 0.2 Da for common ions and 0.3 Da for crosslink ions were used. False discovery rates (FDR) of crosslinked peptides were assigned using xProphet (37). Only high-


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confidence crosslinked peptides that were identified with a delta score (deltaS) below 0.95, a linear discriminant score (xQuestS) above 30, an assigned FDR below 0.05 and that had been detected in multiple, independent experiments, were selected for this study (37). All intermolecular crosslinks were further analyzed by visual inspection in order to ensure good matches of ion series on both crosslinked peptide chains for the most abundant peaks. Visualization of 3D structures and image rendering were performed using VMD (51).

Supporting information available: This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENTS S.D. and E.-M.K. are Postdoctoral Researchers, and V.R. is Senior Research Associates for the National Fund for Scientific Research (FNRS, Belgium). V.R. acknowledges funding from the FNRS and Van Buuren Foundation. F.S. is a Sir Henry Wellcome Fellow (Wellcome Trust Grant 095951). R.A. acknowledges funding from ETH Zurich and ERC grant Proteomics v3.0 (233226). A.M. and N.H. thank the FRIA (Fonds pour la Recherche dans l’Industrie et l’Agriculture) and E.H. thanks the FWO (Research Foundation Flanders) for a doctoral fellowship. We acknowledge M. Prévost for helpful discussions and careful reading of the manuscript.



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Figures and Table

Figure 1. Comparative analysis of the influence of apoE2/3/4 on A fibril formation. Morphology of A alone (A), apoE2/3/4:A at a 2:1 molar ratio (B) and apoE2/3/4 alone (C) imaged by transmission electron microscopy after incubation for 24h at 37°C and staining with uranyl acetate. Without apoE, well-formed A fibrils are observed. In the apoE2/3/4:A and apoE2/3/4 samples only small structures are visible.


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Figure 2. [(apoE)1(A)1] complex formation. Western blot of apoE2/3/4 (as indicated on top of the lanes) in the presence of A, mixed at a 2:1 molar ratio, after incubation for 24h at 37°C. Samples were run on a 4−12% gradient SDS-Page under reducing conditions and detected with anti-A antibody. Blot was saturated for better visualization. SDS-resistant complex between monomer of A and apoE2/3/4 is clearly visible above 35 kDa.



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Figure 3. ApoE:A complex formation with disulfide-bonded apoE isoforms E2 and E3. Western blots of apoE2/3/4 (as indicated on top of the lanes) in the presence of A, incubated at a 2:1 molar ratio for 24h at 37°C. Samples were run on a 4−12% gradient SDS-Page under non-reducing conditions and detected with anti-apoE (A) or anti-A antibodies. Blots were saturated for better visualization. SDS-resistant apoE:A complexes are formed whether apoE2/3 are disulfide-bonded or not.


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Figure 4. Crosslinking of apoE2/3/4 and A. Incubation of monomeric A with apoE2/3/4 at a molar ratio of 1:2 for 24h at 37°C and crosslinked with 4 molar equivalents of bis[sulfosuccinimidyl]suberate (BS3) over apoE. Samples were run on a 4−12% gradient SDS-Page under reducing conditions. Western blot was detected with anti-A antibody. Upon crosslinking, the intensity of the SDS-resistant [(apoE)1(A)] complex at 40 kDa is enhanced.



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Figure 5. CX-MS results on apoE:A binding interface. (A) Schematic representation of apoE:A intermolecular crosslinks detected by MS analysis. ApoE helices are colored according to their domain (NTD, in orange; hinge region, in green; CTD, in blue). A -sheets are colored in red. Helices and sheets are drawn proportionally to their number of residues. Crosslinks identified are shown with dashed black lines and numbers indicate the involved apoE lysine. (B-D) Mapping of the apoE lysines involved in intermolecular apoE:A crosslinks to human apoE3 high resolution structure obtained by NMR (PDB 2L7B) (30). Structures are color-coded as in (A) and crosslinked Lys residues are shown as purple sticks. (B) Intermolecular crosslinked apoE Lys residues on apoE3 NTD, (C) at the apoE3 NTD/CTD interface and (D) on full-length apoE3.


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Table 1. DSS intermolecular apoE:A crosslinks identified for isoforms E2, E3 and E4. Crosslinked lysines

ApoE isoforms

ApoE

A

E2

E3

E4

Lys72 Lys75 Lys95 Lys143 Lys157

Lys16 Lys16 Lys16 Lys16 Lys16

√ ─ √ √ √

√ ─ √ √ √

√ √ √ √ √

Lys242

Lys16

√

√

√



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80x39mm (300 x 300 DPI)


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Mass Spectrometry Maps ... - ACS Publications

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