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Articles Cite This: ACS Chem. Biol. 2019, 14, 1573−1582

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Familial L723P Mutation Can Shift the Distribution between the Alternative APP Transmembrane Domain Cleavage Cascades by Local Unfolding of the Ε‑Cleavage Site Suggesting a Straightforward Mechanism of Alzheimer’s Disease Pathogenesis Eduard V. Bocharov,*,†,‡ Kirill D. Nadezhdin,†,‡ Anatoly S. Urban,†,‡ Pavel E. Volynsky,† Konstantin V. Pavlov,§ Roman G. Efremov,†,‡,∥ Alexander S. Arseniev,†,‡ and Olga V. Bocharova†,‡ Downloaded via BUFFALO STATE on July 24, 2019 at 06:09:50 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Shemyakin−Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, 117198, Russian Federation Moscow Institute of Physics and Technology, Dolgoprudnyi, 141701, Russian Federation § Federal Clinical Center of Physical-Chemical Medicine of FMBA, Moscow, 119435, Russian Federation ∥ National Research University Higher School of Economics, Moscow, 101000, Russian Federation ‡

S Supporting Information *

ABSTRACT: Alzheimer’s disease is an age-related pathology associated with accumulation of amyloid-β peptides, products of enzymatic cleavage of amyloid-β precursor protein (APP) by secretases. Several familial mutations causing early onset of the disease have been identified in the APP transmembrane (TM) domain. The mutations influence production of amyloid-β, but the molecular mechanisms of this effect are unclear. The “Australian” (L723P) mutation located in the Ctermini of APP TM domain is associated with autosomaldominant, early onset Alzheimer’s disease. Herein, we describe the impact of familial L723P mutation on the structural-dynamic behavior of APP TM domain studied by high-resolution NMR in membrane-mimicking micelles and augmented by molecular dynamics simulations in explicit lipid bilayer. We found L723P mutation to cause local unfolding of the C-terminal turn of the APP TM domain helix and increase its accessibility to water required for cleavage of the protein backbone by γ-secretase in the ε-site, thus switching between alternative (“pathogenic” and “non-pathogenic”) cleavage cascades. These findings suggest a straightforward mechanism of the pathogenesis associated with this mutation, and are of generic import for understanding the molecular-level events associated with APP sequential proteolysis resulting in accumulation of the pathogenic forms of amyloid-β. Moreover, age-related onset of Alzheimer’s disease can be explained by a similar mechanism, where the effect of mutation is emulated by the impact of local environmental factors, such as oxidative stress and/or membrane lipid composition. Knowledge of the mechanisms regulating generation of amyloidogenic peptides of different lengths is essential for development of novel treatment strategies of the Alzheimer’s disease.

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and in vivo. Amyloid-β (Aβ) peptides (forming plaques in brain during AD) and related P3 peptides are the products of sequential paramembrane and intramembrane cleavage of a single-span membrane amyloid precursor protein (APP) by α-, β-, and γ-secretases.4,5 Most of the APP mutations associated with familial forms of early onset AD were found in the APP transmembrane (TM) domain and juxtamembrane (JM) regions.6,7 The pathogenic mutations presumably affect structural-dynamic properties of the APP TM domain, e.g., changing its conformational stability, lateral dimerization, and intermolecular interactions, which can result in enhanced and alternative cleavage by γ-secretase in membrane.8−15

lzheimer’s disease (AD) is the most common cause of neurocognitive disorders and may contribute to 60−70% of cases of dementia (according to the WHO report). It is known to induce memory loss, decline in problem solving capability, language, motor skills, and other central neural system functions. Despite some progress in understanding the molecular mechanisms of AD development, the initial steps of the pathogenesis are still puzzling. Though the disease is usually associated with advanced age, sometimes patients are diagnosed with the disease in their forties, or even earlier. Approximately 10−15% of the cases of early onset Alzheimer’s (diagnosed before the age of 65) are familial AD,1−3 where genetic predisposition leads to pathological states. Whenever a genetic anomaly causing early onset Alzheimer’s can be credibly attributed to a single mutation, it provides a promising tool for studying various aspects of the disorder both in vitro © 2019 American Chemical Society

Received: April 18, 2019 Accepted: June 10, 2019 Published: June 10, 2019 1573

DOI: 10.1021/acschembio.9b00309 ACS Chem. Biol. 2019, 14, 1573−1582

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Figure 1. NMR spectra of the L723P-APPjmtm mutant peptide. Two-dimensional (2D) 1H/15N-HSQC NMR spectra of the L723P-APPjmtm mutant peptide (top panels) and the 1H projections of the spectra (bottom panels) are shown for the fresh sample (left panels) and the same sample after 1 week storage (right panels). Gradual migration of amide group signals from the typical α-helical area into the one corresponding to β-sheet structure19 is clearly visible (the emerging β-sheet pattern is highlighted in gray).

recombinant fragment L723P-APPjmtm with the “Australian” mutation obtained as described in ref 18 and solubilized in an aqueous suspension of dodecylphosphocholine (DPC) micelles under monomeric conditions13 with the peptide/ detergent ratio of 1/150, 318 K, pH 6.9, was investigated by means of heteronuclear NMR spectroscopy. The heteronuclear NMR 1H/15N-HSQC spectra (Figure 1) revealed that the overall fold of the mutant fragment in the micelles is identical to that of the wild-type fragment, including the nascent JM helix Gln686-Val695 and TM helix Lys699Lys724.13,17 On the time scale of weeks, this conformation of the mutant fragment proved unstable, gradually evolving from a typical α-helical structure toward a β-sheet conformation (Figure 1) (according to the characteristic chemical shift distribution).19 This process is accompanied by formation of higher molecular weight aggregates, which were initially sufficiently small to be resolved in the high resolution NMR spectra (less than ∼100 kDa), but in the longer run grew to the point of precipitation. In order to find out whether the oligomeric β-aggregates are fibrillar or not, we carried out thioflavin-T fluorescence assay for the NMR sample, which is the classical fibril detection method using the fibril-associated dye.20 The fluorescence was about the same as in freshly prepared probes and negative control (empty micelles only, data not shown), proving absence of amyloid fibrillar structures. Besides that, according to our data at the peptide/detergent ratio of 1/110, the pattern of chemical shift changes of the mutant upon dimerization is similar to that of the wild-type APPjmtm (Supporting Information (SI) Figure S1), implying that the dimerization interface remains unchanged. The only difference is that formation of the dimer is followed by rapid oligomerization, which suggests that the

Several of the familial mutations are located in the APP TM domain, but outside of the Aβ amino acid sequence, and are believed to modulate the process of sequential proteolytic cleavage of APP by γ-secretase. Eventually, this causes a change in the Aβ42/Aβ40 ratio. For example, the “French” V715 M and “German” V715A mutations within the APP TM domain are reported to enhance accessibility of the γ-secretase to εcleavage site and consequently increase Aβ42/Aβ40 ratio.11 The aim of this work is to study another, “Australian” missense L723P mutation located in the C-terminus of the APP TM domain. It increases Aβ42(43) production by a factor of 1.4− 1.9 and causes cell apoptosis.16 Interestingly, the mutation is located beyond the range of the γ-secretase cleavage sites, approximately at the level of the intracellular phase separation boundary of the plasma membrane. The mutation-induced change of the TM domain position in the membrane was implicated in causing alteration of the γ-secretase activity.16 Herein, we provide direct experimental justification for this inference supported by molecular modeling simulations, and offer a putative mechanism explaining the effect of the mutation on the γ-secretase cleavage process.

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RESULTS Comparison of Conformational Dynamics of the Wild-Type and Mutated APP TM Fragments Using High-Resolution NMR in Micellar Environment. Previously, we investigated a wild-type recombinant APPjmtm peptide corresponding to the APP686−726 fragment (Aβ15−55 in amyloid-β numeration) including the intact TM domain with the adjacent N-terminal JM region (without cation-binding domain of Aβ) using heteronuclear NMR spectroscopy in a membrane mimetic environment.13,17 In the present work, a 1574

DOI: 10.1021/acschembio.9b00309 ACS Chem. Biol. 2019, 14, 1573−1582

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Figure 2. Comparison of the wild-type and mutant peptide NMR spectra. Assigned NMR spectra of the wild-type APPjmtm peptide (A) and its L723P mutant (B) and the amide group generalized chemical shift differences between the wild-type and the mutant (C) are shown. The 2D spectra are overlaid with the CLEANEX spectra (indicative of the chemical exchange the amide groups with water, i.e., water accessibility of the amides) shown in red. The measurement uncertainty of the generalized chemical shift differences is shown in the upper right corner. Dual amino acid residue numbering is provided in panel (C), corresponding to the full length APP (black) and Aβ peptides (blue). The alternative γ-secretase cleavage cascade sites leading to production of the ostensibly more pathogenic Aβ42 and less pathogenic Aβ40 peptides are shown by red and green arrows, respectively. The dark green arrow shows the α-secretase cleavage site. Schematics of the secondary structure deduced from our own data13,17 and structures published by other groups23−25 is provided at the bottom. The helical region with enhanced internal dynamics is highlighted in gray.

spatially extended perturbation of the C-terminal part of the TM helix (starting from the residue T719). In addition to that, the structure of the C-terminus of the JM helix, and, to a lesser degree, the N-terminal parts of both TM and JM helices were also affected despite the flexible loop region connecting the TM and JM helices. A summary of the structural-dynamic information obtained from the NMR spectra is provided in Figure 3 (see also SI Figure S2). In order to better understand the subtle perturbations introduced into the peptide structure and dynamics by the mutation, we measured rotational correlation times (τR) characterizing ps−ns internal motions for backbone amide protons along the entire sequence of the wild-type APPjmtm and its mutant L723P form (Figure 3C). According to the obtained data, the C-terminal part of the peptide (3 residues before and 3 residues after L723P mutation) is more flexible in the mutant form: τR values are decreased by 0.5−2.5 ns for the C-terminal residue, as visualized in Figure 3F. Besides that, the τR values are indicative of increased stability in the nascent JM helix and the central part (near the helixdestabilizing diglycine insert G708G709) of the TM helix of the mutant. Consistently with these findings, analysis of the proton−proton nuclear Overhauser effect (NOE) connectivities, as well as the secondary structure probabilities (SSP) and local order parameters S2 derived from 1H, 13C, and 15N chemical shifts (capturing motions at ps−ns and possibly

dimers nucleate formation of oligomers of the mutant fragment (for that reason, in our experiments we used the peptide/ detergent ratio of 1/150 where virtually no dimerization occurs). The lack of long-term stability, or in other words the intrinsic conformational lability of the L723P-APPjmtm secondary structure, is a characteristic trait of mature Aβ peptides. By contrast with this mutation, the same fragment with several other naturally occurring familial Alzheimer’s mutations in the TM domain and JM regions (e.g., “London” V717I/G, “Arctic” E693G, and “Iowa” D694N mutations; data not published yet) was found to be stable on the time scale of a week. As a side note, the slow transition from α-helix to βstructure makes the L723P-APPjmtm peptide incorporated into micelles (and possibly other membrane mimetics) a convenient model to study nucleation of conformational rearrangements associated with amyloidogenesis. The observed loss of long-term stability implies that the conformational energy landscape of the mutant fragment is different from that of the wild-type, implying that secondary structure of the mutant is more susceptible to external stimuli and can readily change in response to alterations of the external conditions. Figure 2 illustrates the comparison between the wild-type and mutant peptide spectra with the amide proton assignments and changes of the generalized chemical shifts of amide protons induced by the mutation. As seen from the chemical shift difference, the mutation caused a pronounced and 1575

DOI: 10.1021/acschembio.9b00309 ACS Chem. Biol. 2019, 14, 1573−1582

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Figure 3. Comparison of the generalized structure-dynamic information for the wild-type APPjmtm peptide and its L723P mutant. (A) and (B). The secondary structure probabilities (SSP) and local order parameters S2 derived from 1H, 13C, and 15N chemical shifts for the given residue of the wild-type (black bars) and L723P mutant (orange and green bars) peptides are indicative of the helical structure distribution and its stability along the sequence of the peptides (see also SI Figure S2). (C) 15N-relaxation of the amide groups of wild-type (black bars) and L723P mutant (lime bars) peptides is characterized by local rotation correlation times τR estimated from 15N CSA/dipolar cross-correlated transverse relaxation. Smaller relaxation times correspond to higher backbone flexibility. (D) H/D exchange rates of slowly exchanging backbone amide protons of wild-type (black bars) and L723P mutant (cyan bars) peptides. (E) Accessibility of backbone amide protons of wild-type (black bars) and L723P mutant (blue bars) peptides to water (W-exchange) assessed using CLEANEX experiment. (F) Visual summary of the most characteristic patterns of changes of chemical shifts (orange), mobility (green and lime), and water accessibility (cyan and blue) induced by L723P mutation. The yellow arrows show the direction of presumable displacement of the polypeptide chain relative to the surfaces of membrane leaflets.

the hydrophobic core of the micelle. Moreover, as can be seen in Figure 3E,F, several residues of the JM region lose accessibility to bulk water, which can be attributable to their insertion into the hydrophobic core of the micelles. In order to detect slower processes of exchange with water, we conducted hydrogen−deuterium (H/D) exchange experiments (see Figure 3D,F; SI Figure S2) that revealed higher overall stability of the wild-type structure compared to the mutant on the large time scale (from minutes to days), which correlates with the tendency of the mutant to slowly convert into βstructure. The observed drastic increase (by a factor of 10 or higher, for residues starting from I718) of the H/D exchange rates of the backbone amide groups in the C-terminal part of the mutant is consistent with its unfolding, and the resulting increase of water accessibility of the C-terminal residues can indeed facilitate the γ-secretase attack. In addition to that, an interesting observation can be made in relation to the Nterminal half of the TM helix. Overall, the H/D exchange rate is higher for both peptides because of enhanced water accessibility of the glycine-rich sequence G700/G704/G708G709,

higher time scales) unambiguously show that the C-terminal part of the TM domain of the L723P mutant is not in helical conformation (Figure 3A,B,F; SI Figure S2). This can facilitate access of water molecules to the unfolded part (the last turn) of the TM helix, which can upregulate the γ-secretase cleavage of the protein in the ε-site location (Figure 3F). On the Nterminal side of L723P-APPjmtm certain (dynamic) folding occurs, most likely due to strengthening of interaction of the JM region with the micelle surface. In order to test this hypothesis, we evaluated water accessibility of the amide groups of residues along the sequence of the peptide. We performed CLEANEX NMR experiments to find the HN-protons that are undergoing rapid exchange with water (W-exchange), and found K724 to be more accessible to water in the L723P mutant than in the wild-type (see Figures 2 and 3E,F; SI Figure S2). It is interesting to note that the residues in the first turn of the mutant TM helix (K699, G700, and A701) are less accessible to water compared to the same residues of the wild-type. This could be explained by deeper immersion of the N-terminal part of the TM helix into 1576

DOI: 10.1021/acschembio.9b00309 ACS Chem. Biol. 2019, 14, 1573−1582

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Figure 4. Molecular dynamics simulations for the wild-type and mutant APP TM fragments in explicit lipid bilayer. MD simulations for the wildtype and the mutant peptide in the explicit POPC bilayer for two protonation states of E693 and D694 residues corresponding to the acidic and neutral pH values: (A) wild-type, protonated; (B) wild-type, unprotonated; (C) mutant, protonated; (D) mutant, unprotonated. The topmost panels provide color-coded representation of the time evolution of the peptide secondary structure (blue, α-helix; gray, 310-helix; yellow, turn; green, bend; white, coil). The bottom maps (second row) show the time evolution of TM helix bending, which is color-coded according to local bending angle from white (0°) to red (20°). Intermolecular contacts (the third row − with the lipid polar headgroups; the fourth row − with water molecules) are color-coded according to the number of direct van der Waals contacts between atoms with 4 Å distance cutoff from white (no contacts) to black (20 protein−lipid or 50 protein−water contacts). The bottom panels are the corresponding representative MD snapshots of the peptides in the POPC bilayer (only phosphorus atoms of the headgroups are shown by orange spheres). The peptides are given in ribbon presentation, glycine and alanine residues are shown in green, L723 in violet, P723 in red, and the rest of the sequence in yellow. The red sphere in the flexible hinge region (with the red arrow pointing to it) in the (B) column represents a water molecule. N- and C-termini of the TM helical segment are indicated.

Molecular Dynamics of the Wild-Type and Mutated APP TM Fragments in Explicit Lipid Bilayer. The αsecretase cleavage occurs primarily on the cell surface in the bulk lipid phase of the plasma membranes, whereas β- and γsecretase proteolyses occur primarily in lipid rafts, most often following internalization of the protein into cholesterol-rich acidic endosomes.5,21 In recognition of this difference, we carried out MD simulations for two protonation states of E693 and D694 residues corresponding to the acidic and neutral pH values, for both the wild-type and the mutant peptide. Figure 4 illustrates evolution of the secondary structure and intermolecular interactions (protein−lipid and protein−water contacts) along 500 ns MD traces. The traces obtained for the wild-type peptide in the protonated and deprotonated

where there are no bulky side chains and the helix surface is slightly polar (in addition to the H/D exchange rate in the Nterminus of any helix being generally higher due to partial positive charge induced by the helix dipole). However, the difference between the H/D exchange rates in the wild-type APPjmtm and its mutant L723P form is much smaller (30− 40%) compared to that of the C-terminal residues, implying that the helical structure in this region is preserved. Moreover, residue M706 of the mutant has a smaller H/D exchange rate, suggesting that the mutation results in burial of this residue into the membrane, thus making it less accessible to water. This is consistent with our interpretation of CLEANEX results that revealed a slight displacement of the N-terminal residues of the mutant toward the membrane interior (Figure 3F). 1577

DOI: 10.1021/acschembio.9b00309 ACS Chem. Biol. 2019, 14, 1573−1582

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TM α-helix seen as a change of the NOE patterns and the chemical shifts, which result in decrease of the helical structure probabilities and local order parameters S2 (Figure 3A,B; SI Figure S2). These changes would not only make the ε-site more readily accessible for cleavage, but also slightly extend and shift the unfolded part of the C-terminal helix toward the γ-secretase active site near the cytoplasmic membrane surface, thus increasing the cleavage rate and potentially changing the likelihoods of initiation of alternative cleavage cascades (i.e., stepwise reduction of the resulting Aβ-peptide length as 48 > 45 > 42 vs 49 > 46 > 43 > 40). More specifically, the Aβ42/ Aβ40 ratio can increase due to the alternative cleavage pathway (starting from the ε-site position ε48 rather than ε49, as indicated by red and green arrows in Figure 2C) becoming more preferable for the mutant because transient unfolding in the case of the mutant extends up to residue T719 (T48 in Aβ numbering). Besides that, the overall shift of the TM segment of the peptide toward the cytoplasmic leaflet of the membrane (shown by an arrow in Figure 3F), which can be inferred from the decrease of water accessibility of several residues on the Nterminal side reciprocated by a matching increase on the Cterminal side of the peptide, is beneficial for productive presentation of the ε-site to the catalytic center of the enzyme. Such a shift should similarly contribute to redistribution of the cleavage products in favor of Aβ42. On the other hand, partial folding of the JM region and its deeper immersion into the membrane experimentally observed for the mutant peptide can interfere with α-secretase cleavage, thus increasing the relative output of Aβ. Hence, both effects of the familial mutation on the protein structure and dynamics tend to increase the yield of the potentially pathogenic APP cleavage products. For better understanding of the pathophysiological implications of these changes, we conducted a series of MD simulations for the wild-type and mutant peptide in the conditions of the NMR experiments with neutral pH corresponding to the peptide in the plasma membrane, and in the acidic environment, to which the peptide is normally exposed in vivo in the endosomes.5,21 MD analysis of the secondary structure behavior (Figure 4) corroborates the findings of the solution and solid state NMR in micelles and liposomes concerning the augmented relative (as compared with the wild-type peptide) flexibility and distortion of the helical structure in the hinge region elongated slightly toward the C-terminus of the APP TM helix from the membrane midplane (previously described as a region from the helixdestabilizing diglycine insert G708G709 to approximately V717),8,13,17,24,25 where the γ- and ξ-cleavage sites are situated. As evidenced by the plots showing distribution of water− peptide contacts along the MD traces, this site is capable of holding water molecules within the hydrophobic membrane core, the bend in the hinge providing a possibility for increased shielding of the molecule from the hydrophobic environment. The effects of mutation upon the overall secondary structure (Figure 4C,D) proved to be essentially similar to the acidification effects observed for the wild-type protein (Figure 4A,B), though the mutation effects are considerably more pronounced. In comparison with the wild-type peptide in the neutral conditions, both the mutation and acidification cause unfolding of the last turn on the C-terminal side of the peptide, the consequential shortening of the APP TM helix compensated by straightening of the hinge region, and partial insertion of the N-terminal JM loop region between the JM and TM helices into the membrane. The nascent JM helix

forms differ dramatically. The most interesting, and presumably physiologically relevant, differences occur on the Cterminus and in the TM helix hinge region (roughly between the helix-destabilizing diglycine insert G708G709 and V717), which is almost permanently bent and unfolded under normal pH and becomes straightened and much more stable upon acidification. The C-terminal region is always unfolded to a certain degree, but in the acidic conditions the unfolding goes further along the peptide sequence (maximally up to V721 at neutral pH and to L720 at low pH), and the lifetime of the unfolded state gets much larger. Moreover, folding of the TM helix hinge (∼100 ns on Figure 4A) results (with a gradual transient process lasting several tens of nanoseconds) in unfolding of the C-terminal region. Under the neutral pH, the correlation, though not as strong as in the acidic conditions, is still clearly noticeable. Apparently, the structure of the flexible water-exposed JM region is also affected by protonation, with a slight shift of conformational distribution toward 310-helical structure and somewhat deeper snorkeling under the membrane lipid heads (based on protein−lipid and protein− water contacts) in the protonated state. The evolution of the secondary structure shown in Figure 4C,D illustrates that the mutation disfavors the kinked (bent) configuration of the central hinge region, the effect being especially pronounced for the neutral pH values. Straightening of the hinge allows the TM domain to adapt to a moderate shortening of its helical portion (from 699−72313,17 to 699− 720) induced by the mutation through destabilization of the helical structure on the C-terminus. On the other hand, loss of the helical structure at the end of the TM fragment is accompanied by increased stability of the JM region, which is generally more helical (α- and 310-helices) in acidic than in neutral conditions. These interpretations of the secondary structure evolutions are corroborated by the analysis of MD data on contacts of the peptide’s amino acid residues with water and with the lipid polar headgroups (Figure 4). In the presence of the kink in the hinge region within the hydrophobic core of the membrane, water molecules can be transiently stabilized in this area (Figure 4A,B), whereas straightening of the hinge abolishes water accessibility. At the same time, the mutation appears to cause an overall shift of the TM fragments toward the cytoplasmic leaflet, as evidenced by an increase of contact with water and lipid polar heads of a few residues on the C-terminal side reciprocated by a matching decrease of such contacts on the N-terminus (Figure 4C,D). Finally, the JM interaction with the membrane surface is notably strengthened by the mutation, especially in the acidic conditions.

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DISCUSSION Enzymatic cleavage of APP by γ-secretase is believed to play a definitive role in determining the spectrum and the distribution of the Aβ-peptide lengths, whereas α- and β-secretase cleavage primarily defines the presence of the cation binding region in the product peptide. The γ-secretase is not known to have exclusive specificity toward a unique peptide bond, cleavage of which it catalyzes; on the other hand, destabilization of the TM helical structure of the substrate peptide near the cleavage site appears to be required for effective cleavage.8,12,22,23 In the APP TM segment, the most visible structural-dynamic changes caused by the L723P mutation are a decrease of the protein mobility in the hinge region evidenced by the observed increase of τR and destabilization of the C-terminal turn of the 1578

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Figure 5. Model illustrating the relations between the structuraldynamic properties of the APP JM-TM segment and AD development induced by mutations and local alterations of the environment. (A) Unrestricted mobility of the central hinge region (in cyan) favors “normal” APP cleavage cascades. (B) Structural-dynamic changes in the hinge and the helix-terminal regions associated with L723P mutation (and possibly some other TM mutations) cause early AD onset, shifting the balance between the cleavage cascades. (C) Local acidification on the plasma membrane surface (e.g., under the oxidative stress conditions) and/or violations of lipid homeostasis (e.g., cholesterol imbalance) more frequently occurring in the advanced age can induce changes similar to the effect of L723P mutation, triggering the age-related AD. The effects of the environmental conditions and familial mutations (or polymorphisms) are not independent of each other, and can be both cooperative (as in case of acidification and cholesterol enrichment) and compensatory (antioxidants or polyunsaturated lipids, as viable candidates), accelerating or delaying AD onset. As before, the yellow arrows show the direction of presumable displacement of the polypeptide chain relative to the surfaces of membrane leaflets, green arrows designate “normal” processes, and red arrows their pathological variants.

APP TM domain. The variations in the relative accessibility of the residues around the membrane boundary to water and lipid headgroups associated with the L723P mutation and with protonation of ionogenic residues in the extracellular JM region are concordant with the relatively mild pathogenicity of the “Australian” mutation (average onset ∼ the age of 56) and age-related increase of exposure to acidic conditions (oxidative stress marker) (Figure 5B,C). Besides the point mutations that result in “straightening” or readjusting the hinge region (e.g., the “Australian”, and probably the “French” and “German” mutations), the stability of the kinked configuration can be affected by APP dimerization and/or by interactions with other membrane components, including certain lipids. For example, interactions of the N-terminal part of the TM helix and the adjacent extracellular loop region with cholesterol favor the “sunk” position of the amphiphilic JM region,17,24 potentiating the effects of protonation of ionogenic residues in the latter, e.g., under oxidative stress conditions (Figure 5C). Moreover, the presence of polyunsaturated lipids in the immediate vicinity of the APP can modulate the effects of cholesterol,21,26 emphasizing a potential role of overall lipid homeostasis in the AD onset and progression. More importantly, local lipid environment can prevent Aβ production by affecting the hinge configuration and relative position of the α-helix distortion with respect to the membrane surface, e.g., shifting it toward the cytoplasmic leaflet, where the ε-site of cleavage by γsecretase is located. The recently published cryo-EM structure27 of γ-secretase-APP complex (with C-terminal APP-C83 TM fragment) stabilized by cross-linking at the APP JM region suggests that the N-terminal part of the TM fragment and the hinge region are accessible for interaction 1579

DOI: 10.1021/acschembio.9b00309 ACS Chem. Biol. 2019, 14, 1573−1582

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ACS Chemical Biology 1

H/15N-HSQC (heteronuclear single quantum coherence spectroscopy), 1H/13C-HSQC, 1H/13C-HSQC-CT (constant time version with evolution period of 28.6 ms), 1H/15N-TROSY (transverse relaxation optimized spectroscopy), 1H/15N-HNHA, 1H/13C/15NH N C A , 1 H/ 1 3 C / 1 5 N- HN(CO)CA, 1 H/ 1 3 C / 1 5 N -HN CO, 1 H/13C/15N-HN(CA)CO, 1H/13C-HCCH-TOCSY (with the mixing time of 17 ms), 13C- and 15N-edited NOESY-HSQC (with the mixing time of 80 ms). Spectra were recorded with nonuniform sampling of indirect dimensions and processed using the qMDD software.32 The backbone resonances of the peptides were assigned using the BESTTROSY version of the triple resonance experiments31 based on the APPjmtm chemical shifts previously obtained at pH 6.2. 13 Generalized chemical shift differences, Δδ(15NH), for the amide groups (Figure 2C) are calculated as the geometrical distance (with weighting of 1H shifts by a factor of 5 compared to 15N shifts) between the amide cross-peaks assigned to the residues of L723PAPPjmtm and APPjmtm. The secondary structure probabilities (SSP) and local order parameters S2 were estimated from the 1H, 13C, and 15 N chemical shift values using the web-based program TALOS-N.33 Intramolecular 1H−1H NOE connectivities were obtained with the CARA software through the analysis of the three-dimensional 15Nedited NOESY-HSQC spectrum. In order to characterize the intramolecular dynamics of the APP TM fragments, the effective rotation correlation times τR were estimated for individual amide groups of the fragments based on 15N CSA/dipolar cross-correlated transverse relaxation experiment acquired in interleaved fashion for the reference and attenuated spectra using a 2D 1H/15N-ct-TROSYHSQC-based pulse sequence34 with the constant period of 26.9 ms and the relaxation period of 10.8 ms. The rotation correlation times τR was calculated as the ratio of peak intensities of the reference and attenuated spectra according to the equation from ref 34, the uncertainties were estimated from the noise level. The wateraccessibility of the APPjmtm and L723P-APPjmtm residues was analyzed by detection of chemical exchange of the amide protons with water detected by the CLEANEX experiment35 at 318 K. For measuring the exchange rates between water and HN protons, we reconstituted lyophilized samples of APPjmtm and L723P-APPjmtm in D2O and within 1 week acquired a series of 25 1H/15N-HSQC spectra. The peak intensities as functions of time were fitted by monoexponential equations; the uncertainties were estimated as standard deviations of the fitting. The H/D exchange experiments were made at the same pH of 6.9, but the temperature had to be decreased to 303 K in order to decelerate the exchange to the detectable rates. Molecular Dynamics in Explicit Lipid Bilayer. In order to assess conformational dynamics and intermolecular interactions of the APP686−726 fragment and its mutant L723P form in explicit lipid bilayer, molecular dynamics (MD) simulations were performed using GROMACS 5.1.4 package36 and Amber99SB-ILDN force-field3037 with TIP3P water model38 and lipid parameters as described elsewhere.39 The initial monomeric conformations of the wild-type peptide and its mutant L723P form were derived from the NMR structure of the APPjmtm dimer13 (PDB ID: 2loh). Protonated and charged states of ionogenic side chains of the E693 and D694 residues were used to emulate acidic and neutral pH conditions. The starting configurations of the simulated systems were obtained by inserting of the APP TM fragments into a pre-equilibrated lipid bilayer comprised 200 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) molecules. The systems were solvated and necessary counterions were added to make the system electrically neutral. The latter were equilibrated by 1000 steps of steepest descent energy minimization followed by 250 ps MD runs with fixed protein atoms. Finally, 500 ns MD runs were carried out for all the systems using a 2 fs time integration step. The spherical cutoff function (12 Å) was used for truncation of van der Waals interactions, while electrostatic effects were treated using the particle-mesh Ewald summation (real space cutoff 10 and 1.2 Å grid with fourth-order spline interpolation). MD simulations were carried out with imposed 3D periodic boundary conditions in the isothermal−isobaric (NPT) ensemble with the semiisotropic pressure of 1.013 bar scaled independently along the bilayer

with the neighboring membrane lipids, whereas the C-terminal part is in the extended conformation starting from T719 residue exposing the γ-secretase cleavage sites to the enzyme active center. The fact that the familial L723P mutation causes the helix to constitutively unwind in the same region thus supports our conclusion that the mutation prepares the APP TM segment for effective attack by the γ-secretase at both T719/L720 and L720/V721 cleavage sites (ε48 and ε49 in Aβ numbering), resulting in production of Aβ42 and Aβ40 peptides, respectively. According to our data, in the wild-type protein such unfolding of the C-terminal part occurs only transiently and is less extended into the membrane by one or two residues, which should result in a smaller overall production of Aβ and a smaller share of pathogenic Aβ42 form. Besides that, the cryoEM structure contains a small-angle (partially straightened) kink in the same position as described for the free APP TM domain.17,27 It is to be expected that without the S−S bond linking APP JM region to γ-secretase there would be a higher degree of mobility in the hinge region,28 and depending on the external factors, to which this part of the γ-secretase-APP complex remains exposed (pH, local lipid composition), the hinge can assume different configurations favoring diverse cleavage pathways. This can imply that for individuals without AD mutations, after the age of 65 (the age-related AD), the combined impact of persistent oxidative stress caused by overall metabolism downregulation and of altering lipid metabolism can result in a shift of distribution between the APP cleavage cascades similar to that caused by L723P mutation.

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CONCLUSIONS In summary, we have demonstrated with the aid of NMR and MD methods that on the molecular level familial AD L723P mutation causes local unfolding of the C-terminal turn of APP TM domain helix and increases its accessibility to water required for cleavage of the protein backbone by γ-secretase in the ε-site. Such a change appears to be sufficient to cause switching between the alternative (“pathogenic” and “nonpathogenic”) cleavage cascades. These findings suggest a straightforward mechanism of the pathogenesis associated with this mutation as well as other AD familial mutations found in the APP, and are of generic import for understanding of the molecular-level events associated with the APP sequential proteolysis resulting in accumulation of the pathogenic forms of amyloid-β.

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METHODS

NMR Spectroscopy in Micellar Environment. The 15N/13Clabeled recombinant peptide APPjmtm (MQ686KLVFFAEDVGSNKG 7 0 0 AIIGLMVGGVVIATVIVITLVML 7 2 3 KKK 7 2 6 , fragment APP686−726, hydrophobic TM segment is underlined) and its mutant L723P form (L723P-APPjmtm) were expressed and purified according to ref 18. The APP TM fragments were solubilized in an aqueous suspension of d38-dodecylphosphocholine (d38-DPC, 98%, CIL) micelles under monomeric conditions with the peptide/ detergent ratio of 1/150, 20 mM NaPi buffer, pH 6.9, 5% D2O (v/ v), as described in refs 13, 18. High-resolution heteronuclear NMR spectra of the 0.4 mM APPjmtm and L723P-APPjmtm samples were acquired at 318 K on 600 and 800 MHz AVANCE III spectrometers (Bruker BioSpin) equipped with pulsed-field gradient triple-resonance cryoprobes. The 1H, 13C, and 15N chemical shifts of APPjmtm and L723PAPPjmtm at pH 6.9 were assigned with the CARA software29 by means of two- and three-dimensional heteronuclear experiments:30 1580

DOI: 10.1021/acschembio.9b00309 ACS Chem. Biol. 2019, 14, 1573−1582

ACS Chemical Biology

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normal and in the bilayer plane at a constant temperature of 310 K. The pressure and temperature were controlled using Nose-Hoover thermostat and Parrinello−Rahman barostat with 0.5 and 10 ps relaxation parameters, respectively, and a compressibility of 4.5 × 10−5 bar−1 for the barostat. Temperatures of protein, lipids, and water molecules were coupled separately. Analysis of conformational dynamics of a protein and its van der Waals contacts with lipid and water molecules was performed using the GROMACS package utilities. In order to map the protein−lipid and protein−water interactions, the numbers of direct van der Waals contacts between atoms within 4 Å distance were estimated. Local bending angle of the APP TM helix was calculated for the region Lys699-Met723 using the Bendix plugin of the VMD package.40

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ABBREVIATIONS AD, Alzheimer’s disease; APP, amyloid precursor protein; Aβ, amyloid-β; TM, transmembrane; DPC, dodecylphosphocholine; NMR, nuclear magnetic resonance; MD, molecular dynamics.

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ASSOCIATED CONTENT

* Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.9b00309. Detailed description of some NMR data obtained for the APP TM fragments in membrane mimetic (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Eduard V. Bocharov: 0000-0002-3635-1609 Roman G. Efremov: 0000-0002-5474-4721 Author Contributions

EVB and OVB conceived and designed experiments, interpreted the results and wrote the manuscript (approved by all authors). ASU and OVB obtained the peptides and prepared the NMR samples. EVB, ASU, and KDN conducted and analyzed the NMR experiments. PEV conducted and analyzed the MD experiments. EVB, ASU, KDN, and PEV generated and edited the figures. KVP and KDN helped to interpret the results and write the manuscript. RGE and ASA discussed the results and provided critical input on the manuscript. Funding

NMR and bioengineering work was supported by the Russian Foundation for Basic Research (projects #17−04−02045 and #18−54−74001). MD simulations and computational data analysis of the APP TM fragments in explicit bilayer were sponsored by Russian Science Foundation (project #18−14− 00375). Supercomputer calculations were supported in the framework of the Russian Academic Excellence Project “5− 100”. Notes

The authors declare no competing financial interest.

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REFERENCES

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ACKNOWLEDGMENTS

The authors express their sincere thanks to K.A. Beirit for helpful discussions. Experiments were partially carried out using the equipment provided by the IBCH core facility (CKP IBCH, supported by Russian Ministry of Education and Science, grant RFMEFI62117X0018). Access to computational facilities of the Supercomputer Center “Polytechnical” at the St. Petersburg Polytechnic University and Joint Supercomputer Center RAS (Moscow) is greatly appreciated. 1581

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DOI: 10.1021/acschembio.9b00309 ACS Chem. Biol. 2019, 14, 1573−1582