Formation of extramembrane β-strands controls dimerization of transmembrane helices in amyloid precursor protein C99

Abstract

The 99-residue C-terminal domain of amyloid precursor protein (APP-C99), precursor to amyloid beta (Aβ), is a transmembrane (TM) protein containing intrinsically disordered N- and C-terminal extramembrane domains. Using molecular dynamics (MD) simulations, we show that the structural ensemble of the C99 monomer is best described in terms of thousands of states. The C99 monomer has a propensity to form β-strand in the C-terminal extramembrane domain, which explains the slow spin relaxation times observed in paramagnetic probe NMR experiments. Surprisingly, homodimerization of C99 not only narrows the conformational ensemble from thousands to a few states through the formation of metastable β-strands in extramembrane domains but also stabilizes extramembrane α-helices. The extramembrane domain structure is observed to dramatically impact the homodimerization motif, resulting in the modification of TM domain conformations. Our study provides an atomic-level structural basis for communication between the extramembrane domains of the C99 protein and TM homodimer formation. This finding could serve as a general model for understanding the influence of disordered extramembrane domains on TM protein structure.

Keywords: amyloid; intrinsically disordered; lipid bilayer; molecular dynamics; protein–protein interaction.

Fig. 1.

Left y-axis and bar plots: I/I₀ is the ratio of full-length C99 1H, 15N backbone amide NMR signals in the presence of either hydrophilic (Gd-DTPA) or hydrophobic (16-DSA) paramagnetic probes over peak intensities observed in probe-free conditions. Right y-axis and scatter plots: The observed likelihood of C99 monomer and homodimer backbone amide or carbonyl hydrogen bond formation with water in gREST simulations at an effective temperature of T = 310 K. Green dashed lines indicate bilayer surface position. The L_d phase Gd-DTPA NMR experiment was performed with 100 μM uniformly 15N-enriched monomeric C99 in 10 wt% in solution q = 0.33 POPC/DDMB bicelles with 1 mM Gd-DTPA. L_o phase Gd-DTPA and 16-DSA experiments performed with 200 to 400 μM uniformly enriched partially multimeric C99 in 5 to 10 wt% in q = 0.33 4:2:1 DMPC:eSM:Chol/DDMB bicelles and in the presence of either 6 to 9 mol% 16-DSA (relative to the total moles of lipid) or 1 mM Gd-DTPA in solution (31).

Fig. 2.

Analysis of the full-length C99 monomer in hydrated L_d phase bilayers at 310 K. (A) Silhouette scores for Ward’s minimum variance hierarchical clustering (inset dendrogram) quantify the quality of assignments of configurations to k number of clusters. The dashed red line is in the elbow of S(k) at k = 350. (B) Percent of conformational ensemble for each cluster, p(cluster) (black), ranked in order from largest to smallest, and the cumulative sum of p(cluster) (red). (C) First largest cluster, β-sheets in yellow. TMD in blue and POPC phosphorous in green. (D) Ensemble-averaged secondary structure of monomer ensemble assigned using STRIDE showing random coil propensities in the N-terminal extramembrane domain and β-strand propensities in the C-terminal extramembrane domain also suggested by NMR probe solvent accessibility in Fig. 1 (31). (E) Secondary structure of conformations in which the C-terminal helix is present. (F) 5th largest cluster, C-terminal helix in red.

Fig. 3.

Analysis of the full-length C99 homodimer in hydrated L_d phase bilayers at 310 K. (A) Silhouette scores for Ward’s minimum variance hierarchical clustering (inset dendrogram) for partitioning of C99 homodimer conformations into k number of clusters. The dashed black line identifies the optimal number of clusters at k = 15. (B) Ensemble average and population-ranked cluster secondary structures assigned using STRIDE show many unique β-strand motifs in the N- and C-terminal extramembrane domains. (C) Representative conformations of 1st, 2nd, and 13th largest clusters. TMD in blue, C-terminal helix in red, and POPC phosphorous and Gly33 Cα in green.

Fig. 4.

Differences in ensemble-averaged structure of monomeric and homodimeric C99. Intraprotein backbone N-H⋯O=C hydrogen bonds for monomer (A) and dimer (B) demonstrate formation of more stable intraprotein hydrogen bonds in extramembrane domains. (C) Ensemble average secondary structures of monomeric C99, homodimeric C99, and the change in secondary structure upon homodimerization show introduction of N-terminal β-strands, C-terminal juxtamembrane β-strands, and C-terminal extramembrane α-helices upon homodimerization.

Fig. 5.

Monomeric (A) and homodimeric (B) C99 TM domain tilt angle θ and Gly33-Gly34 hinge angle κ demonstrate a slight stiffening in the Gly-Gly hinge and a broader TM tilt upon homodimerization. (C) Dihedral of Gly29–Gly33–Gly33–Gly29 in the full-length homodimer, defining a left- (ϕ > 0) and right-handed (ϕ < 0) superhelix. (D) Secondary structure of C99 for Gly-side (12th and 13th-largest clusters), Gly-out (2nd-largest cluster), and Gly-out (the remaining clusters), and secondary structure for left- and right-handed superhelices. (E) Gly33–Gly33 Crick angles in the xy-plane, colored by conformations belonging to left- and right-handed superhelices. Inset homodimer conformation from 13th-largest cluster shows a Gly-side Ψ ∼ (90 ° ,150 ° ) Crick angle configuration. (F) Lys28–Lys28 and Lys54–Lys54 Cα pair distances. Only left-handed superhelices (blue) access X- and Y-shaped homodimer motifs.

Fig. 6.

Interprotein backbone hydrogen bonds within 2.5 Å hydrogen distance and ≥ 150 angle between the C99 carbonyl oxygen acceptor and (A) Cα–hydrogen donor and (B) amide hydrogen donor. Solid gray lines specify the beginning of the TM domain from residues 28 to 55. Dashed blue lines identify TM glycine residues 29, 33, 37, and 38. The lack of Cα–hydrogen bonding in the TM domain involving the glycine zipper and frequently observed N- and C-terminal hydrogen bonds suggests that these extramembrane domain interactions stabilize the dimer.

Fig. 7.

Homodimer extramembrane residues 1 to 28 and 55 to 99 average secondary structure propensity as a function of (A) Gly33–Gly33 Crick angles and (B) Lys28–Lys28, Lys54–Lys54 Cα pair distances demonstrate the unique dimer interfaces induced by promotion of α helices in extramembrane domains, particularly formation of the juxtamembrane helix in the 2nd-largest cluster.