New Results

Structure of APP-C991-99 and Implications for Role of Extra-Membrane Domains in Function and Oligomerization

George A. Pantelopulos, John E. Straub, D. Thirumalai, Yuji Sugita
doi: https://doi.org/10.1101/257469

Abstract

The 99 amino acid C-terminal fragment of Amyloid Precursor Protein APP-C99 (C99) is cleaved by γ-secretase to form Aβ peptide, which plays a critical role in the etiology of Alzheimer’s Disease (AD). The structure of C99 consists of a single transmembrane domain flanked by intra and intercellular domains. While the structure of the transmembrane domain has been well characterized, little is known about the structure of the flanking domains and their role in C99 processing by γ-secretase. To gain insight into the structure of full-length C99, REMD simulations were performed for monomeric C99 in model membranes of varying thickness. We find equilibrium ensembles of C99 from simulation agree with experimentally-inferred residue insertion depths and protein backbone chemical shifts. In thin membranes, the transmembrane domain structure is correlated with extra-membrane structural states. Mean and variance of the transmembrane and G37G38 hinge angles are found to increase with thinning membrane. The N-terminus of C99 forms β-strands that may seed aggregation of Aβ on the membrane surface, promoting amyloid formation. The N-terminus, which forms α-helices that interact with the nicastrin domain of γ-secretase. The C-terminus of C99 becomes more α-helical as the membrane thickens, forming structures that may be suitable for binding by cytoplasmic proteins, while C-terminal residues essential to cytotoxic function become α-helical as the membrane thins. The heterogeneous but discrete extra-membrane domain states analyzed here open the path to new investigations of the role of C99 structure and membrane in amyloidogenesis.

Introduction

Amyloid Precursor Protein (APP), a 770-residue membrane protein, plays important roles in neural activity and regulation of synaptic formation.[1] The canonical APP processing pathway is defined by APP cleavage by either by α- or β- secretase resulting in 83-or 99-residue long peptides (C83 and C99), that form the majority of APP fragments in cells.[2] C99 can subsequently undergo processive cleavage of its transmembrane domain by γ-secretase at various sites within its transmembrane (TM) region, yielding 38, 40, 42, and 43-residue long N-terminal fragments commonly known as Amyloid β (Aβ) protein.[3] Aβ42 (and to some extent Aβ43) has been implicated in the onset of Alzheimer’s disease (AD) due to the presence of fibrillar aggregates enriched in these peptides[4,5] found in the brains of AD patients.[6] In addition, Aβ42 oligomers have been directly observed to accompany loss of neural plasticity and memory.[7]

Solution NMR measurements[7,8] employing zwitterionic bicelles and micelles provide the primary source of information on the structure of C99 in a variety of membrane mimicking environments. In these in vitro environments, there is evidence that residues 1-14 (see Figure 1) of the N-terminal domain (NTD) are disordered, residues 15-25 of the N-terminus have helical propensity (N-Helix), residues 26-28 form a turn (N-Turn), residues 29-52 form the helical transmembrane domain (TMD), residues 53-90 of the C-terminus form a disordered region (C-Loop), and residues 91-99 of the C-terminus form a helix (C-Helix).[8,9] Insertion of residues in the membrane evidenced by EPR[8] and NMR[9] measurements suggest that in some systems the C-Helix and N-Helix domains rest on the membrane surface, while the proximities of the NTD and C-Loop domain to the membrane remain unclear.

Figure 1.
Figure 1.

Yellow shading represents hydrophobic core of the membrane. Red residues contain familial AD mutations. Blue residues are critical to the formation of C99 dimers. Green residues may be phosphorylated. Purple residues form the lysine anchor. Black residues indicate γ-secretase cleavage sites and metal binding residues. Brown residues are critical for C31 formation and cytotoxicity. Cylinders represent domains with significant helical propensity. θ and κ angles describe the TMD tilt and GG hinge angle. The θ angle increases with thinning or curving of the membrane surface, and κ increases with curving of the membrane surface. Black solid lines mark the membrane surface and black dashed lines represent the membrane hydrophobic core. The orange lipid marks the putative cholesterol binding site. Also shown is an atomistic structure of C99 predicted from TALOS+ using LMPG micelle backbone chemical shifts and secondary structure assigned with STRIDE. Cα within the atomistic structure are labeled as N-terminal familial AD mutation (red), residues 28, 37, 38, 53,54, and 55 (orange), phosphorylatable residues Ca (green), and C-Helix (blue).

The structure of the TMD is believed to be critical to the mechanism of recognition and cleavage of C99 by γ-secretase. The process of cleavage of C99 by γ-secretase begins with the “ε-cleavage” step, forming Aβ48 or Aβ49, which are then further cleaved via “ζ-cleavage” to form Aβ45 and Aβ46. These fragments are subsequently processed by “γ-cleavage” to predominantly form Aβ38 or Aβ42, and Aβ40 or Aβ43, respectively.[10] C99 features a glycine zipper motif, G29xxxG33xxxG37, in the TMD, which is frequently observed in dimer-prone single-pass TM proteins.[11,12] It is further evidenced to be a component of putative cholesterol binding site on C99,[13,14] a finding that is important because cholesterol has been hypothesized to recruit C99 to γ-secretase.[14–16] Mutation of G29 and G33 in this motif reduces Aβ42 production,[17] and is expected to reduce C99 dimerization.[18] Proximate to the N-terminal portion of the GxxxG repeat motif lies a “GG hinge” at G37G38 in the TMD, previously identified by molecular dynamics simulations[19,20] and conjectured to be important to processing by γ-secretase.[21] Hydrogen-deuterium (H-D) exchange studies observed side chain[21] and a-helix[22] hydrogen bonds to be substantially weaker near the GG hinge, suggesting the amide bonds are readily available for γ-cleavage. Thickening of the membrane reduces the relative amount of Aβ42 and Aβ43 produced while leading to an overall increase in γ-secretase activity.[23,24] Increasing the curvature of membrane is found to increase the magnitude of fluctuation of the GG hinge and the overall tilt of the TMD.[25] It is likely that magnitude of fluctuations in the hinge may enhance Aβ42 and Aβ43 production.[8] Additionally, simulation studies have revealed[20,26,27] that the GG hinge is an important structural feature for C99 dimers, with the angle of the hinge varying for several distinct dimerization motifs. It has further been noted that the membrane thickness can preferentially stabilize and environmentally select specific C99 dimer strucutres.[17,26–28] Beyond the hinge lies G38xxxA42, another glycine zipper motif often found in TM dimers,[18] important for C99 homodimerization.[29] The GxxxG repeat motif appears to facilitate C99 dimer formation in thicker membranes while the competing GxxxA motif supports dimers are most often observed in thinner membrane and micelle.[30] At the C-terminal end of the TMD, residues A42, T43, V44, I45, V46, T48, L52, and K53 all feature several mutations found in AD.[31] Some mutations decrease the propensity for homodimerization,[32] and enhance Aβ42 production.[33] A “lysine anchor” formed by the tripe repeat K53K54K55 is evidenced to register at the C-terminal end of the TMD membrane surface.[34]

While the TMD structure has been the focus of experimental and computational studies, the structure of the extra-membrane residues has received relatively little attention in spite of the suspicion that the extra-membrane domains of C99 have interesting features that are crucial to AD. The N-terminus of C99 almost certainly interacts with the nicastrin domain of γ-secretase.[35] Within the N-Loop domain, Ala point mutation of K28 has a dramatic impact on APP processing, switching formation of Aβ40 to Aβ33, implicating this turn in the γ-secretase interaction.[34] Residues 15 to 21 (LVFFAED of the N-Helix domain), sometimes referred to as the juxta-membrane (JM) region, is plays a role in inhibiting γ-secretase binding[36] and binding with cholesterol.[8,13,15] Furthermore, membrane insertion of residues in the JM region appears to sensitively depend on pH.[13] The N-Helix also features mutants A21G,[37] E22Q,[38] E22K,[39] E22G,[40] E22Δ,[41] and D23N,[42] all found to occur in AD patients. Within the NTD, the mutation K16N is known to make APP untenable for binding by α-secretase[43,44] and the E11K mutation was found to enhance Aβ production.[45] The mutations D7H,[46] D7N,[47] H6R,[47] and A2V[48] were found in patients with early onset of AD, suggesting a role for these residues in interaction with γ-secretase. Additionally, histidine residues in the N-terminus H6, H13, and H14 are known to bind with Cu and Zn metals, found in high concentration in amyloid plaques.[49] Additionally, Aβ42 forms a complex with the C99 N-terminus when C99 is membrane-bound, which enhances C99 homo-oligomer formation.[50]

In the C-Loop there are several phosphorylatable residues, identified at T58, S59, T72,[46] and Y86.[51] The phosphorylation of S59 enhances trafficking of APP to the golgi apparatus.[52] It has been noted that Ala point mutation at T72 may enhance the production of Aβ40 and Aβ42,[53–55] impacting interaction of APP with some enzymes.[56] Y86 has been identified to be phosphorylated at higher concentrations in the brains of AD patients, and is suspected to prevent the interaction of APP with adaptor proteins.[54]

The C-Loop and C-Helix are known to interact with several proteins in the cytoplasm, forming complexes in which these domains adopt an a-helical structure.[57] The C99 sequence binds to many cytoplasmic proteins including the G protein G0 with residues H61-K80,[58] the adaptor protein Fe65 with residues D68-N99,[59] the adaptor protein X11 with residues Q83-Q96,[60] the adaptor protein mDab1 with a similar residues to X11,[61] and the kinase Jip-1with residues N84-F93.[62] The C-terminus is cleaved by caspases at D68 to form C31, a cytoplasmic protein found in AD patients and evidenced to signal apoptosis.[63] Aβ-C99 complex-enhanced C99 oligomerization increases the production of C31.[50] Mutation of D68 to Ala prevents production of C31, abrogating cytotoxic function.[64] Additionally, residues 85-91 (GYENPTY) are found to be essential for cytotoxic activity of C31, and are involved in interactions with many cytoplasmic proteins.[50]

Currently, the experimental knowledge of extra-membrane residues of C99 is limited to backbone chemical shifts, NOEs, and EPR signals from membrane-mimicking environments, for which the structural ensembles of residues 6, 12-16, 53-56, 62, 73-76, 80, 81, and 88 are unresolved or too uncertain.[8,9] A prodigious body of work characterizing structure of Aβ fragments has been performed and generally suggests that residues 21-28 of Aβ act as a seed for oligomerization and fibril formation. It has been conjectured that this region contains key residues characterizing the aggregation-prone N* state of Aβ and Aβ fragments.[65,66] Support for this conjecture has been provided by NMR and computational studies of Aβ40 and Aβ42 structural ensembles.[67] Additionally, the N-Turn and C-Loop domains show nearly random coil chemical shifts, implying that they are unstructured on average. However, these domains may exhibit heterogeneity of metastable structural states as has been observed in many intrinsically disordered proteins. The current knowledge of C99 structure and residue features in typical thermodynamic conditions is summarized in Figure 1.

To address some of the outstanding questions related to the structure of C99 and its interaction with the membrane we performed simulations of monomeric wildtype C99 in model membranes, using a computational approach that proved to be remarkably useful in elucidating structures of the TMD.[19,20,27] We employed replica-exchange molecular dynamics (REMD)[68] to sample hundreds of nanoseconds of C99 dynamics at physiological temperatures in 30, 35, and 40 Å-thick membranes modeled using the GBSW implicit solvation method.[69]

In thin membranes we observed extracellular domain states to be correlated with the TMD state. The mean and variance of the TMD and GG hinge angles (Figure 1) were observed to increase with thinning of the membrane. The C-and N-terminal secondary and tertiary structures of C99 were heterogeneous with discrete metastable states. These ensembles were directly compared and contrasted with the results of prior solution NMR and EPR studies. C99 ensembles were found to exhibit newly-observed metastable α-helical and β-strand structures in C- and N-termini, which are correlated with the state of the TMD only in thin membranes. β-strand structures observed in some N-terminal residues are suggestive of templates that may seed amyloid oligomerization on the membrane surface. α-helical domains in the N-terminus are observed and found to be suggestive of nicastrin association sites. α-helical domains observed in previously uncharacterized phosphorylatable sites T58, S59, and Y86 (see Figure 1), suggest that these domains may be involved in interactions that enhance phosphorylation processes. Overall, our work provides an enhanced picture of the structure of extra-membrane residues of C99, and lays the foundation for further investigations of considering the role of C99 structure in facilitating interaction with other molecules in membrane.

Methods

A. Initial Structure Preparation

We constructed an initial structure of the full-length C99 sequence using current literature data. Residues 23-55 were modelled using a “Gly-in” structure of one C99 sampled in the recent work by Dominguez et. al.[27] Onto this fragment, residues 1-22 and 56-99 were built using dihedral angles predicted via the TALOS+,[70] using the Cα, Cβ, C, N, and H chemical shifts reported for C99 in LMPG micelles.[30] To remove clashes and effectively move the C-Helix close to the membrane surface, the ψ angle of H14, located in the disordered loop of the N-teminus (see Figure 1), was adjusted to 180° and the φ angle of Q82 was adjusted to 180°. The rotomeric states of residues 1-22 and 56-99 were assigned using the Shapovalov and Dunbrack rotamer library.[71] Protonation states were assigned using the AddH program in UCSF Chimera,[72] assigning negative GLU and ASP, positive LYS and ARG, neutral CYS and TYR, and setting HIS to the HSD CHARMM histidine type. The center of the membrane was initially set at the z-coordinate of the Cα of G38.

B. Molecular Dynamics Simulation

All simulations were performed using CHARMM version c41b1[73] using the CHARMM36 force field,[74] likely to be the most accurate force field for simulation of Aβ.[75] The GBSW implicit membrane solvent model was used,[69] employing a 0.004 kcal mol-1 Å-2 surface tension, 5 Å smoothing length from the membrane core-surface boundary, and 0.6 Å smoothing length at the water-membrane surface boundary, using 24 radial and 38 angular integration points to 20 Å. No cutoffs were used for nonbonded interactions. After C99 was inserted in 30, 35, and 40 Å-thick implicit membranes the potential energy was minimized using the steepest descent algorithm until apparent convergence, and simulated for 130, 160, and 460 ns, respectively, using REMD[68] via the REPDSTR utility in CHARMM. We used 16 replicas in REMD simulations, employing exponentially-spaced temperatures from 310 to 500 K and attempting to exchange temperature conditions every 1 ps, manifesting an overall exchange success rate of 17.7 ± 3.7%. Langevin dynamics was employed using a 2 fs time step with a leap frog integrator, a 5 ps-1 friction constant, and constrained hydrogen bonds via the SHAKE algorithm. Atomic coordinates were written every 10 ps and all analyses employed coordinate data at this resolution.

C. Analysis Methods

SciPy,[76,77] Cython,[78] Matplotlib,[79] VMD v1.9.3,[80] MDAnalysis v.0.16.2,[81,82] MDtraj v1.9.0[83] were employed for analyses. SHIFTX2 was used to compute the full set of chemical shifts of C99 in each frame for thermodynamic conditions of 7.4 pH and 310 K temperature.[84] All analyses considered structures sampled at equilibrium (past 30 ns) in the 310 K ensemble from REMD. To assign conformational states of extra-membrane domains of C99, a relatively low-dimensional space that enables precise clustering was constructed. Secondary structure assignments were made using the STRIDE implementation in VMD.

To cluster C99 structures four Principal Component Analysis (PCA) eigenspaces were constructed, using Ca positions and the sine and cosine transformations of dihedral angles (dPCA[85]) of N-terminal residues 1-29 and C-terminal residues 52-99, using data from the equilibrium ensemble in 30, 35, and 40 Å membranes. The first 3 principal components of simulation data in each of these four eigenspaces were considered relevant, each conformation of C99 described by a 12-dimensional space capturing the secondary and tertiary structure of the N-and C-terminus. Conformations at each membrane thickness were assigned to states by clustering in this 12-dimensional space using a 16-cluster Gaussian Mixture Model (GMM). The GMM of simulations at each membrane thickness was constructed using k-means clustering to parameterize initial clustering and weights of each data point in each cluster, then refined using 100 iterations of the GMM Expectation-Maximization algorithm.[86] Metric multidimensional scaling of the 12-dimensional data to 2 dimensions for each membrane thickness was performed in order to visualize the nature of the clustering and the relative distance between states.

Results and Discussion

A. Convergence of Ensemble to Equilibrium and Experiment

Full C99 sequence was simulated using REMD in GBSW implicit membranes, a successful approach for enhanced sampling of membrane protein structure.[87] Membranes of 30, 35, and 40 Å thicknesses, corresponding to lipids such as DLPC, DMPC, and POPC, were used to study the effect of membrane structure on the conformational ensemble of C99.[88] The initial structure of C99, constructed from a combination of past simulations and chemical shift-based dihedral assignments (Figure 1), gradually evolved in REMD simulations to interact with the membrane surface. The radius of gyration (Rg) rapidly converged to the ensemble average in 35 and 40 Å membranes, but appeared to require 20 ns to converge in 30 Å membranes due to relatively slow re-arrangements in secondary structure near the membrane surface (Figure 2A), evidenced by deep insertion of C99 side chains to the membrane (Figure S1). We considered the equilibrium ensemble to have been reached by 30 ns in all REMD simulations, and only consider data at equilibrium for characterization of C99 structure.

Figure 2
Figure 2

(A) Squared difference of Rg from ensemble average over time. The vertical dashed line at 30 ns demarcates the time beyond which the ensembles are considered to be at equilibrium. (B) Equilibrium average and standard deviation of insertion depth of residue Cα in the membrane, Dins. Stars indicate scaled relative depths of residue insertion to the membrane inferred from EPR probe signals in POPG:POPC membranes.[8] Scaled NMR signals from lipophilic (blue) and hydrophilic (cyan) probes in POPC-DHPC bicelles shown in bars.[9] (C) Equilibrium average and standard deviation of Ca chemical shifts predicted using SHIFTX2. Stars indicate the Ca chemical shifts measured in LMPG micelles. (D) Structures of C99 at 30 ns in 30, 35, and 40 Å membranes. (Same residue coloring as in Figure 1).

The ensemble average of Cα residue depths of insertion (Dins) in the membrane were well-captured by simulation, comparing well with NMR signals from hydrophobic and hydrophilic probes in POPC-DHPC bicelles and correlating with past EPR measurements in POPG:POPC membranes by Pearson’s r of 0.888, 0.861, and 0.894 for 30, 35, and 40 Å membranes, respectively (Figure 2B). This marginally higher insertion depth correlation observed in 40 Å membrane may be attributed to the insertion of the C-Helix in the membrane surface. The whole sequence of the C-Helix was observed to rest on the membrane surface in much of the 40 Å ensemble in contrast with the 30 and 35 Å ensembles that predominantly show residues around T90 to rest on the membrane surface. The higher correlation of C99 residue insertion in 40 Å implicit membranes is coincident with POPC membrane, which has been measured to be approximately 40 Å thick in combined analysis of small-angle neutron and X-ray scattering data.[88]

Cα chemical shifts predicted using the SHIFTX2 algorithm, which boasts the best correlations of predicted chemical shifts to experiment of current chemical shift prediction methods, show substantial correlation with those measured in LMPG micelles (r correlation coefficients of 0.882, 0.908, and 0.903 for 30, 35, and 40 Å membranes) (Figure 2C). However, overall the 40 Å membrane simulations showed higher correlation with all backbone chemical shifts (Figure S1 and Table S1).[30]

B. TMD tilt and kink angles

The hinge located at G37G38 has been conjectured to modify the interaction of C99 with γ-secretase in a way that impacts C99 processing.[21] As the membrane thickness increases the production of Aβ has been reported to increase overall, but the ratio of Aβ42/Aβ40 decreases.[23,24] This suggests that C99 structures in thick membranes are preferable for appropriate interactions of C99 and γ-secretase. The stability of the TMD helix at the GG hinge has been observed to be weaker than the rest of the TMD via H-D exchange experiments.[21,22]

In past simulation studies the GG hinge flexibility did not appear to be sensitive to membrane thickness.[27] However, the simulations presented here include the full C99 sequence, which seems to be important for sampling certain TMD structures (Figure 3). Here, we define the TMD tilt angle (θ) as the angle between the vector of best fit through residue 30-52 Cα positions and the z-axis (Figure 1). The GG hinge angle (κ) is the angle between the vectors of best fit through Ca positions of residues 30-37 and of residues 38-52 (Figure 1). In 40 Å membranes there is a single macrostate of TMD structure with average and standard deviation in TMD angles (<θ>) of 7.5° ± 3.9° and GG hinge angles (<κ>) of 9.3° ± 4.9°. In 35 Å membranes these angles increase to <θ> = 11.1° ± 5.6° and <κ> = 9.9° ± 5.1°. In 30 Å membranes three structural macrostates of the TMD manifest, composing 29.8% (TM1), 66.2% (TM2), and 4.0% (TM3) of the ensemble. Extramembrane clusters 4, 5, 7, and 8 comprise TM1, featuring <θ> of 9.1° ± 3.6° and <κ> of 16.6° ± 7.0°. Clusters 1, 2, 3, 6, 9, 10, 12, 13, 14, 15, and 16 comprise TM2, which exhibits <θ> of 24.3° ±4.0° and <κ> of 14.5° ± 7.0°. Cluster 10 comprises TM3, characterized by <θ> of 9.3° ±4.3° and <κ> of 46.9° ± 4.8°. The extreme kink in TM3 is an artifact, resulting from unraveling of TMD residues 31-33 to form a β-strand with residues 20-22. These observations suggest that the mean and variance of TMD and GG hinge angles generally increase as a result of membrane thinning. The increase is accompanied by considerable heterogeneity in the C99 structures.

Figure 3.
Figure 3.

TMD (θ) and GG kink κ angles of C99 (A) PMF (-kBT ln(p)) at equilibrium in 30, 35, and 40 Å membranes, showing 10,000 randomly selected data points in red and (B) 30 Å membrane for TMD macrostates TM1 (blue), TM2 (red), and TM3 (gold), which compose 66.2, 29.8, and 4% of the equilibrium ensemble, respectively. Insets show mean and standard deviation of angles in the displayed macrostate. Representative C99 conformation secondary structure drawn with STRIDE and Ca colored as defined in Figure 1.

C. Secondary Structure, Membrane Insertion, and Implications of C99 States

The secondary and tertiary structures of extra-membrane residues are heterogeneous. Using projection of simulation data onto a 12-dimensional space the describing relevant PCA eigenvectors of secondary and tertiary structures of N-and C-terminal extra-membrane residues, conformational clusters were assigned and refined using k-means and a Gaussian Mixture Model to find the 16 conformational states defined in 30, 35, and 40 Å membranes (Figs S3-5). These clusters were inspected by embedding the 12-dimensional space to a 2-dimensional space by metric multidimensional scaling and viewing all assigned atomistic structures (Figs S6-8).

Considering the 8 most populated clusters of each membrane, which account for 75.4, 75.4, and 73.0% of 30, 35, and 40 Å membrane ensembles, we identify the most prominent C99 states. Secondary structure assignment via STRIDE allows for the general classification of structure. We consider the secondary structure propensity by taking the difference between the observed α-helix likelihood (pα) and the observed β-strand likelihood (pβ) (ps = pα - pβ) for each cluster (Figure 4). In each membrane condition, we observe unique secondary structures including or proximal to sites of non-TMD familial AD mutations, phosphorylatable sites, and the metal binding sites. To consider tertiary structure we measured the insertion depth of Cα to the membrane surface (Dins).

Figure 4.
Figure 4.

Difference of α and β propensity at each C99 residue in the ensemble (see scale for secondary structure propensity on the right) and in the 8 most populous clusters in 30, 35, and 40 Å membranes (percentages correspond to population of the ensemble). Lines and text indicate residue indices of interest: AD-associated mutations (red), phosphorylation sites (green), lysine anchor (purple), metal binding sites (black), Aβ33-producing mutation (orange), and C31 cleavage and cytotoxic function sites (brown). Last frame of visualized C99 clusters with secondary structure drawn with STRIDE and Cα shown as in Figure 1.

The TMD is observed to lengthen on both the C- and N-terminal ends with increasing membrane thickness. The TMD was extended by two residues at the N-terminus and one residue at the C-terminus every 5 Å increase in membrane thickness, growing from residues 30-53 in 30 Å membranes to residues 26-55 in 40 Å membranes. This observation is in contrast to the usual assumption[34] that the lysine anchor does not change its registration with the membrane surface, and that only the N-terminal end of the TMD changes registration with the membrane surface as membrane thickness changes. This had previously been unconfirmed, as past experiments on full-length C99 could not resolve structure or membrane insertion of the lysine anchor in a variety of environments.[9] Residue K28, found to change production of Aβ40 to Aβ33 when mutated to Ala,[34] is incorporated in the TMD helix, undergoing a transition from β to α structure as membrane thickness increases.

In all membrane conditions residues 16-20 have helical propensity and to be inserted to the membrane, in agreement with prior EPR and NMR experiments.[34] The full C-Helix, identified as being inserted in membrane in past experiments, is found to be helical in all conditions other than 30 Å, for which residues 96-99 are unstructured and unassociated with the membrane surface. The C-Helix is observed to be helical even when unassociated with the membrane surface, a condition observed in some clusters in all membrane conditions. This finding provides a structural basis for the conjecture that the C-Helix is available for binding with cytoplasmic proteins in any membrane condition. Residues 73-76, for which membrane insertion and chemical shifts had been previously unresolved in experiments, appear to be unstructured in all membranes and broadly distributed relative to the membrane surface. Residues 74 to 80 are found to be slightly less helical and more bound to the membrane surface in 30 Å membranes, suggesting that thinner membranes may make C99 less available for binding to the G0 protein, which binds residues 61-80.[58] Residue D68, the cleavage site for cytotoxic C31 peptide formation, gains more β-propensity as membrane thins. The cytotoxic functional domain G85-Y91 becomes more a-helical in response to membrane thinning, though the insertion depth does not follow a trend, being membrane-associated in 30 and 40 Å, and membrane-disassociated in 35 Å membranes. It may be possible that C99 in thinner membranes is more amenable to cleavage of D68 to form C31.

In 30 Å membranes, residues 21-23, 25-27, and 28-30 occasionally interact to form β-strands, suggestive of the aggregation-prone N* structural motif observed in Aβ fragments.[65,66] This structure is not present in 35 and 40 Å membranes, in which residues 28-30 join the TMD helix. Mutants of residues 21-23 are featured in cases of familial AD and thin membranes are known to cause an increase in the ratio of Aβ42/Aβ40 produced. It is possible that mutations in residues 21-23 stabilize this β-strand, altering the TMD ensemble to resemble the structure observed in 30 Å membranes. Additionally, in some clusters, residue K55 forms H-bonds consistent with β-strand structures involving A69, occasionally including Q82 and Q83 as well.

Figure 5.
Figure 5.

Average membrane insertion of each C99 residue Cα ensemble (see scale for depth of insertion on the right) in the ensemble and in the 8 most populous clusters in 30, 35, and 40 Å membranes (percentages correspond to population of the ensemble). Lines and text indicate residue indices of interest: AD-associated mutations (red), phosphorylation sites (green), lysine anchor (purple), metal binding sites (black), Aβ33-producing mutation (orange), and C31 cleavage and cytotoxic function sites (brown). Last frame of indicated C99 clusters with secondary structure drawn with STRIDE and Cα shown as in Figure 1.

In 35 Å membranes, a prominent β-hairpin is formed with residues 2-5 and 11-15, in which residue N27 sometimes participates via H-bonding. This hairpin is positioned away from the membrane surface. This structure does not appear in membrane-bound α1-42 in similar implicit membrane simulations,[20] and seems to be unique to 35 Å-thick membranes. It is possible that this β-hairpin structure acts as a seed for Aβ oligomerization. C99-seeded Aβ association to the membrane may be much more favorable than pure Aβ mixtures considered in the past,[89] as Aβ is at substantially higher concentration outside the cells than in the membrane. Mutation of residues 2, 11, and 16, found in familial AD, may change the propensity for this β-hairpin to form. Additionally, H6, H13, and H14, residues known to bind metal ions found at high concentration in amyloid plaques,[49] are proximate to the observed β-hairpin. The structure of His-ion complexes found in computational investigations of Aβ1-16 resembles this hairpin structure.[90,91] As such it may be possible that 35 Å membranes are ideal for stabilizing C99 structures that bind metal ions.

In 40 Å membranes there is weaker propensity for β-hairpin formation observed in 35 Å membranes. A strand with residues 2-5 and 11-13 in some clusters, such as 1, 6, and 8, is observed. In clusters 2 and 4, residues 11-15 form an α-helix that is unassociated with the membrane. Along with residues 16-20, this helix may serve as an interaction site with the nicastrin domain of γ-secretase. The formation of these α-helices may serve to enhance the recognition of C99 by γ-secretase as one possible mechanism explaining the observed increase in Aβ processing observed in thicker membranes.

Figure 6.
Figure 6.

Pearson’s r correlation of average Cα depth of insertion in membrane (Dins) and difference in observed α and β structure propensity (ps) in C99 residues 1-28 and 53-99 in the equilibrium ensemble (E) and in each cluster.

The secondary structure propensities and insertion depth of non-TMD residues 1-28 and 52-99 for the whole ensemble and for each cluster reveal that the helicity of extra-membrane residues is not correlated with membrane insertion depth. This observation is contrary to typical expectation that the more hydrophobic membrane environment increases the propensity for helical structure. This is quantified by Pearson’s r correlation of non-TMD residue secondary structure propensity to membrane insertion of Cα, r(ps, Dins), in the ensemble and in clusters (Figure 6). It is indeed possible that this could be a consequence of the simulation model used, and further investigation using explicit solvent simulations with consideration of the disordered protein structure should be pursued.

Conclusions

We performed REMD simulations of full length C99 in model membranes of 30, 35, and 40 Å thicknesses. We observe the TMD and G37G38 hinge angle means and variances to increase as the membrane thins. Multiple TMD states were found to manifested in thin membranes, with which a positive correlation between TMD states with extra-membrane residue states was found. Heterogeneous but discrete structural states observed in the C99 C- and N-terminal extramembrane regions of C99 are found to be unique to the specific membrane condition. Generally, an increase in α and β secondary structure is observed as membrane thickness increases. The TMD helix expands on the N- and C-terminal ends as membrane thickness increases. Residues 21-23, 25-27, and 28-30 form β-strands similar to the aggregation-prone N* motif previously observed in Aβ fragments in 30 Å membranes. In addition, residues 2-5 and 11-15 form a β-hairpin in 35 Å membranes. It is conjectured that these β-strand motifs may serve as seeds for Aβ aggregation on the membrane surface. Residues 11-15 adopt α-helical structures in 40 Å membranes that may promote binding of C99 with the nicastrin domain of γ-secretase to promote non-amyloidogenic processing of C99. α-helical structures are generally stabilized in the C-terminus as membrane thickness increases, and do not require association with the membrane surface. This observation suggests that these domains are readily available to interact with proteins in the cytoplasm. Conversely, residues 85-91, known to be essential to cytotoxic function, become α-helical as the membrane thins. These observations drawn from our simulation study are summarized in Figure 7.

Figure 7.
Figure 7.

Yellow shading represents the hydrophobic environment of the membrane. Secondary structures resulting from membrane thinning (red), membrane thickening (blue), and unique to 35 Å membranes (gold) are transparent. Residue indices are provided to identify regions in which secondary structure is observed. Red residues are found mutated in familial AD, black residues are important for metal binding, green residues are phosphorylatable, orange K28 Ala mutation changes Aβ produced, and brown residues are critical for C31 formation and cytotoxicity. TMD and GG hinge angle means and variance increase with thinning membrane. Black-outlined C-loop β-strands are transient in many membrane conditions.

The insights provided by this study enhance our current understanding of the structural ensemble of full length C99 in membrane and the potential role played by C99 structure in recognition and processing by γ-secretase. Taken together, these results open the path to investigations of the role of C99 structure in interactions with γ-secretase and Aβ, which may lead to new perspectives on the genesis of amyloid in AD.

Acknowledgements

G.A.P. thanks the NSF GRFP for support under NSF Grant No. DGE-1247312, the NSF GROW program, and the 2017 JSPS Postdoctoral Fellowships for Research in Japan Strategic Program. J. E. S. and D. T. acknowledge the generous support of the National Institutes of Health (R01 GM107703). D.T. thanks the Collie-Welch Regents chair (F0019) for generous support. We thank Afra Panahi for discussion regarding phosphorylatable sites on C99 and assistance in preparation of simulations using the REPDSTR utility. The authors acknowledge the Shared Computing Cluster, administered by Boston University’s Research Computing Services, used for many of the MD simulations.

Footnotes

  • ↵* E-mail: straub{at}bu.edu

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Posted February 21, 2018.
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Structure of APP-C991-99 and Implications for Role of Extra-Membrane Domains in Function and Oligomerization
George A. Pantelopulos, John E. Straub, D. Thirumalai, Yuji Sugita
bioRxiv 257469; doi: https://doi.org/10.1101/257469
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