Allosteric Modulation of Intact γ-Secretase Structural Dynamics

Abstract

As a protease complex involved in the cleavage of amyloid precursor proteins that lead to the formation of amyloid β fibrils implicated in Alzheimer's disease, γ-secretase is an important target for developing therapeutics against Alzheimer's disease. γ-secretase is composed of four subunits: nicastrin (NCT) in the extracellular (EC) domain, presenilin-1 (PS1), anterior pharynx defective 1, and presenilin enhancer 2 in the transmembrane (TM) domain. NCT and PS1 play important roles in binding amyloid β precursor proteins and modulating PS1 catalytic activity. Yet, the molecular mechanisms of coupling between substrate/modulator binding and catalytic activity remain to be elucidated. Recent determination of intact human γ-secretase cryo-electron microscopy structure has opened the way for a detailed investigation of the structural dynamics of this complex. Our analysis, based on a membrane-coupled anisotropic network model, reveals two types of NCT motions, bending and twisting, with respect to PS1. These underlie the fluctuations between the "open" and "closed" states of the lid-like NCT with respect to a hydrophilic loop 1 (HL1) on PS1, thus allowing or blocking access of the substrate peptide (EC portion) to HL1 and to the neighboring helix TM2. In addition to this alternating access mechanism, fluctuations in the volume of the PS1 central cavity facilitate the exposure of the catalytic site for substrate cleavage. Druggability simulations show that γ-secretase presents several hot spots for either orthosteric or allosteric inhibition of catalytic activity, consistent with experimental data. In particular, a hinge region at the interface between the EC and TM domains, near the interlobe groove of NCT, emerges as an allo-targeting site that would impact the coupling between HL1/TM2 and the catalytic pocket, opening, to our knowledge, new avenues for structure-based design of novel allosteric modulators of γ-secretase protease activity.

Figure 1

Structure of γ-secretase, its catalytic cavity, and equilibrium fluctuations. (A) γ-secretase in a lipid bilayer, constructed using the coordinate data in PDB: 5FN2 (11) and the lipid molecules from the Orientations of Proteins in Membranes database. The complex is composed of four subunits shown in different colors (PS1, NCT, APH-1, and PEN-2). NCT forms the ectodomain (except for its C-terminal helix, residues 665–698); the other three subunits form the TMD. The lipid bilayer is shown as gray sticks, with the polar heads in red. The lower diagram shows the nine TM helices of PS1 color-coded as in (C), with the catalytic site enclosed in a blue circle. The hydrophilic loop HL1 connecting TM1 and TM2 is labeled. (B) Ribbon diagram of the complex shown upon rotating the structure in (A) by 90°. Glu333, near the substrate-binding site of NCT, is shown in red, enclosed in a black circle; the catalytic site of PS1 is enclosed in a blue circle. The dashed line on NCT separates the large lobe (LL) and small lobe (SL), which form a large surface groove. (C) Top view of PS1. The lower diagram shows the catalytic residues Asp257 and Asp385, and neighboring Met146, Trp165, Met233, and Gly384 (TM helices colored as in top ribbon diagram). (D) Comparison of experimentally observed (blue) and computationally predicted (red) structure factors for the complex resolved by cryo-electron microscopy. The correlation coefficient between the two sets of data is 0.88. (E) Same comparison as in D, in terms of ribbon diagrams color-coded by residue MSFs from experiments and ANM computations.

Figure 2

Bending and twisting motions of NCT with respect to TMD. (A–D) Pairs of conformers sampled during bending (modes 1 and 7) and twisting (modes 2 and 5) of γ-secretase. In each case, two ANM conformers are shown to illustrate the type of conformational fluctuations driven by the indicated mode. We display the membrane in yellow dots, PS1 in cyan, and PS1 TM2, TM3, and TM7 helices in red. The position of Glu333 in NCT, and Asp257 and Asp385 in PS1, are indicated by green spheres. Distances between Asp257 and Glu333 (blue arrow), Asp541 (NCT) and Tyr115 (PS1) (red arrows in A, C, and D), and Glu584 (NCT) and Tyr115 (PS1) (red arrows in B) are shown. Note that the distances depend on the size of ANM motions, which scale with the force constant γ. Here, the distances corresponding to an RMSD of 4 Å with respect to the original (PDB) structure are shown, for each mode direction. MD simulations (Fig 3) indicated that such distance changes take place within microseconds. See Movies S1 and S2 for the animations of the respective modes 1 and 5.

Figure 3

Results from CG MD simulations of γ-secretase. (A) Motions of NCT with respect to PS1 (cyan). Two snapshots at t = 1.6 and 5.5 μs are displayed (from run 1). The distances between Asp257 and Glu333 (blue arrows) and Tyr115 and Asp541 (red arrows) are indicated. See Fig. S3 for more details. (B) Comparison of the MSF profile of NCT residues predicted by the ANM (red curve), observed in CG MD (blue), and deduced from ensemble analysis of five PDB structures (green). Correlations between the three pairs vary in the range 0.86–0.90, as indicated. (C) Same as (B), for PS1. (D) Correlations between the softest (seven) modes predicted by the ANM and those obtained from the PCA of 10 μs MD trajectory (run 1). High correlations are shown in dark red and weak correlations in dark blue. The table lists the pairs that exhibit the highest correlations. (E) Projections of 10,000 frames from 10 μs trajectory onto the ANM mode 1 and PCA mode 1 directions (left), and ANM mode 2 and PCA mode 2 directions (right). PCA mode 1 is equivalent to the bending (mode 1) predicted by ANM, as illustrated in the ribbon diagram, and PCA mode 2 is equivalent to the twisting mode (ANM mode 2). Similar results obtained in CG MD run 2 are presented in Fig. S2.

Figure 4

Mobility profile of γ-secretase and the critical position and dynamics of HL1. (A) Distribution of residue MSFs for the intact γ-secretase in the membrane (NCT portion identical to the ANM curve is shown in red in Fig. 3B). Highest peaks in NCT are labeled, as well as sites serving as anchors or hinges (e.g., E245, F287, and E333), which exhibit small fluctuations in space. (B) Close-up view of key residues along PS1 MSF profile. PS1 loop HL1 (S104–T124) central portion exhibits large movements. The catalytic residues (D257 and D385) are highly stable (minima). (C) Location of HL1 loop residues (shown as red space-filling) and the broken TM6 (orange) of PS1 where D257 is located. All other structural elements are colored as in Fig. 1A. The location of NCT peak residues is indicated by the white circle. (D) Top view of the TMD (colored as in Fig. 1; NCT is removed for visual clarity). HL1 covers a large portion of the EC-facing vestibule of PS1. See Figs. 5 and S4 for more details.

Figure 5

Intersubunit contacts between NCT LL and PS1 HL1 facilitated by the global bending mode. (A) Initial structure of the complex (PDB: 5FN2). (B) Closed form enabled by ANM mode 1, based on an RMSD of 4 Å from the initial structure. Q540, D541, R543, R583, E584, P593, and S611 in NCT, and Y106, R108-D110, Y115, and E120–E123 in PS1 are shown as red (negatively charged), blue (positively charged), or orange (polar) sticks. We note a cluster of interactions involving D121–E123 on HL1 and Q540–R543 at the NCT LL surface groove mouth (encircled). See also Movie S1 and Table S1 for close intersubunit interactions in the closed form favored by mode 1.

Figure 6

Cross correlations between the motions of γ-secretase residues in the membrane environment. (A) Cross correlation map for γ-secretase complex and surrounding lipid bilayer. The entries in the map represent the orientational cross correlations (−1 ≤ C_ij ≤ 1; see the scale on the color-coded bar) between all pairs of nodes (residues or lipid sites) i and j. Red blocks along the diagonal indicate the strongly correlated substructures, and blue regions indicate the anticorrelated (moving in opposite direction) pairs of substructures. Results are based on 10 softest ANM modes. (B) Close-up view of cross correlations within γ-secretase. (C) Network representation of the complex and membrane, colored by the type and strength of correlation with respect to Asp257 (indicated by dashed line in B). Blue and red regions exhibit anticorrelated and correlated movements, respectively, with respect to Asp257; yellow/green regions are uncorrelated. (D) γ-secretase color-coded by cross correlations of residues with Asp257. Strongest anticorrelations are observed at the NCT LL (blue), whereas PS1 and APH-1 form a highly correlated block (red). The NCT SL (residues 34–248 and 651–664) and NCT terminal TM helix (residues 665–698) include hinge sites (yellow/green). (E) Same as in (B), in the absence of membrane. An overall weakening in the cooperativity of subunit movements is observed.

Figure 7

Anticorrelations between EC-exposed ends and the catalytic site observed in ANM modes 3 and 4 accessible to the complex. Cross correlations driven by ANM modes 3 (A) and 4 (C) are displayed, and corresponding respective diagrams (B and D) color-coded by the correlations of all PS1 residues with respect to Asp257. The two catalytic residues (D257 in TM6 and D385 in TM7) are shown in spheres as well as selected residues (e.g., Y106 (HL1), Y115 (HL1), Y240 (TM5), S132 (TM2), and D450 (TM9)) that either exhibit strong anticorrelations (blue) or act as hinge centers (yellow). Y106, Y115, and Y240 are involved in GSM binding, and S132 and D450 in substrate binding.

Figure 8

Asymmetric breathing motion of PS1. (A) A pair of conformers, compact and stretched, sampled during reconfiguration of PS1 along ANM mode 14 of the protease complex-lipid system is shown. The conformers are obtained based on an RMSD of 4 Å each with respect to the initial (PDB) structure. TM2, TM3, and TM7 are colored red. Glu333 in NCT1, and Asp257 and Asp385 in PS1, are shown in green spheres. (B) Close-up view of interhelical distance changes in the two conformers. TM2, TM3, TM6, TM7, and TM9 are colored as in Fig. 1C; other TM helices are cyan. The left diagrams display PS1 from the same perspective as in (A); the right diagrams are rotated around the normal to the membrane plane, to facilitate the visualization of the TM2–TM9 distance. Asp257, Asp385, Ser132 (TM2), Asn190 (TM3), and Asp450 (TM9) are shown in spheres colored by the corresponding TMs. S132–N190 (left) and S132–D450 (right) distances show opposite changes, i.e., an increase in the former is accompanied by a decrease in the latter and vice versa. (C) Catalytic cavity in the respective conformers, with TM2–TM7 shown in space-filling. Side chain positions obtained by energy minimization are shown for selected residues. See also Movie S3.

Figure 9

High-affinity sites identified by druggability simulations of the intact complex. (A and B) Druggable regions of PS1. TM helices are colored as in Fig. 1A. Three druggable sites are observed, labeled as R1–R3, composed each of a cluster of hot spots (in cream/yellow spheres). Y106 and Y115 (HL1); S132 and M146 (TM2); W165 and F177 (TM3); M233 and Y240 (TM5); L248, L250, and D257 (TM6); G384 and D385 (TM7); and L435, F447, and D450 (TM9) are shown as sticks and labeled. (C) Druggable regions on NCT, labeled RA-RE. Druggable hotspots are shown in white. Sites colored blue, ice blue, and purple participate in the hinge regions, and red spheres act as hinge centers. (D) Square fluctuations driven by ANM modes 1 (black line) and 2 (blue line). The hinge regions and residues are marked by bands (blue, ice blue, and purple) and red arrows. Residues of interest at druggable sites are indicated. (E) Zoom-in views of each druggable region in NCT. Hinge residues close to druggable sites are labeled and indicated by yellow arrows. See Table S2 for estimated binding affinities at those sites, and Fig. S8 for additional druggable sites.

Figure 10

Binding of inhibitors (DAPT and BMS-708163) and modulators (E2012 and ST1120) to PS1. (A) Orthosteric site near D257 and D385, also identified as the top-ranking druggable site R1 (in Fig. 9A), binds both inhibitors. Binding pose of each inhibitor is shown in the magnified diagrams on the right. The same set of residues (labeled) coordinate both drugs. (B and C) Binding of modulators E2012 and ST1120 to the allosteric site R2 identified in Fig. 9A. Coordinating residues include Y106 and Y115 (on the EC loop HL1), N135 (TM2), Phe177(TM3), and Y240 (TM5).

Figure 11

Schematic description of a model for peptide binding, repositioning, cleavage, and release modulated by the opening/closure of NCT with respect to the TMD. (A) Schematic diagram of γ-secretase. NCT is composed of two lobes, LL and SL, shown in green and blue separated by hinge 2, and the transmembrane domain (TMD) is in red, with interfacial hinge 1 enabling the relative movement of NCT SL with respect to TMD. NCT fluctuates between open and closed states. (B) Binding of modulator (GSM, dark red) to the allosteric site (cyan) near HL1/TM2 (on PS1) or the interlobe groove (on NCT) stabilizes the closed form and obstructs the access of the substrate peptide (orange, extended from EC to TM region) to its binding site and to the PS1 catalytic site. Blue ball in NCT is substrate-binding site and blue ball in TMD is catalytic site. (C) Suggested binding pose of substrate, attached on top to NCT, and exposed at the bottom to the catalytic site. (D) Sliding of the substrate to expose a new cleavage site to the catalytic region, facilitated by the closure of NCT. Open and closed states may thus help proper positioning of the successive cleavage sites: ε cleavage occurs in open state (C) and γ cleavage in closed state (D). (E) Subsequent opening of NCT permits the release of the Aβ peptide to the EC region.