Table 1.
ApoE isoform-specific mutations.
Fig 1.
Heat capacity curves of ApoE isoforms.
(A) The heat capacity (Cv) curves computed using WHAM on REX/DMD trajectories for ApoE2 (black), ApoE3 (red) and ApoE4 (blue) in the range of 275 to 400 K show intermediates states that appear at different temperatures for each isoform. The position of the first peak (i.e., unfolding of the hydrophobic core of the protein) suggests that ApoE4 is less thermally stable than ApoE2 and ApoE3. (B-D) Cv curves of individual ApoE isoforms including the error bars (shaded grey area). The shaded grey area in panels B-D represents the statistical uncertainty (i.e., the square root of the variance of the specific heat) in the WHAM estimation of heat capacity. Local minima in the curves at temperatures T1, T2, T3, and T4 represent different conformational states of the protein for each ApoE variant.
Fig 2.
Free energy landscapes of ApoE isoforms.
ApoE isoforms’ conformational landscapes derived from PMF as a function of RMSD and Rg of ApoE variants’ N-terminal domains. C-terminal domains are excluded from the analysis to reduce the degeneracy of protein conformational states. (A-C) The free energy landscapes from REX/DMD simulations at T1 (~275 K for all three ApoE isoforms) are isolated in the lowest range of RMSD and Rg suggesting the majority of conformations are close to the native N-terminal domain state. (D-F) At T2 (~321 K, ~318 K, and ~309 K for ApoE2, ApoE3 and ApoE4 respectively) all three variants explore a larger area of the conformational landscape as denoted by the larger RMSD and Rg values. (G-I) At T3 (~340 K, ~338 K, and ~328 K for ApoE2, ApoE3 and ApoE4 respectively) the isoforms transition to their intermediate states. ApoE3 is characterized by both the native and alternate N-terminal domain conformations, while ApoE2 visits only the latter. ApoE4 exhibits a unique, more compact intermediate conformational state as denoted by the smaller range of RMSD and Rg values compared to the two other variants. (J-L) At T4 (~355 K, ~365 K, and ~342 K for ApoE2, ApoE3, and ApoE4, respectively) all three isoforms undergo complete unfolding as inferred by their extended landscapes in the high range of RMSD and Rg values, although ApoE4 also visits the previous conformational states identified at temperature T3. (Note the different scale on x- and y-axes; representative structures are reported in S2 Fig). The color bar represents the relative Helmholtz free energy in kcal/mol.
Fig 3.
Representative structures of ApoE isoforms.
(A) ApoE3 representative structure (i.e., centroid of the most populated cluster) from clustering analysis of the protein conformations extracted from the free energy basin at T1 (~275 K, see Fig 2B). The same compact, native state of the N-terminal helices is observed in all three ApoE isoforms (see S2A–S2C Fig). At T3 (~340 K, ~338 K, and ~328 K for ApoE2, ApoE3 and ApoE4 respectively) the representative structure of the intermediate state for: (B) ApoE2 exhibits an expanded volume of the N-terminal domain due to an increase of the average inter-helical distances; (C) ApoE3 exhibits a pairing of N-terminal helix–1 and helix–4 which separate from helix–2 and helix–3; (D) ApoE4 exhibits a separation of helix–1 from the other three helices. Such conformation represents the identified isoform-specific misfolded intermediate state (inter-residue contacts shown in S6A Fig) The size of the most populated cluster is reported in each panel. For all structures, helix–1 (H1), helix–2 (H2), helix–3 (H3), and helix–4 (H4) are represented in purple, green, blue, and red, cartoon respectively. The rest of the protein is represented in grey cartoon. (The sequence numbers for helices is reported in S1 Fig).
Fig 4.
Temperature-dependent pair-wise inter-residue distances of ApoE isoforms.
(A-C) At T1 (~275 K for all three ApoE isoforms) and (D-F) at T2 (~321 K, ~318 K, and ~309 K for ApoE2, ApoE3 and ApoE4 respectively), all three isoforms exhibits the highest level of inter-residue contacts observed in the REX/DMD simulations, with ApoE4 having the highest density contacts. (G-I) At T3 (~340 K, ~338 K, and ~328 K for ApoE2, ApoE3 and ApoE4 respectively), all three isoforms exhibit a dramatic decrease in density of inter-residue contacts. ApoE4 displays a unique series of contacts (outlined in red) mediating the domain-domain interaction as discussed in the main text. (J-L) At T4 (~355 K, ~365 K, and ~342 K for ApoE2, ApoE3, and ApoE4, respectively), the majority of inter-residue contacts have been lost besides some transient contacts involving the N-terminal helix–4. The upper and lower triangular matrices represent respectively the average and the standard deviation of the pair-wise inter-residue distance in Å. The color bar represents the distance between the centroid computed over the residues’ side chains in Å.