Microsecond molecular dynamics simulations of intrinsically disordered proteins involved in the oxidative stress response

Abstract

Intrinsically disordered proteins (IDPs) are abundant in cells and have central roles in protein-protein interaction networks. Interactions between the IDP Prothymosin alpha (ProTα) and the Neh2 domain of Nuclear factor erythroid 2-related factor 2 (Nrf2), with a common binding partner, Kelch-like ECH-associated protein 1(Keap1), are essential for regulating cellular response to oxidative stress. Misregulation of this pathway can lead to neurodegenerative diseases, premature aging and cancer. In order to understand the mechanisms these two disordered proteins employ to bind to Keap1, we performed extensive 0.5-1.0 microsecond atomistic molecular dynamics (MD) simulations and isothermal titration calorimetry experiments to investigate the structure/dynamics of free-state ProTα and Neh2 and their thermodynamics of bindings. The results show that in their free states, both ProTα and Neh2 have propensities to form bound-state-like β-turn structures but to different extents. We also found that, for both proteins, residues outside the Keap1-binding motifs may play important roles in stabilizing the bound-state-like structures. Based on our findings, we propose that the binding of disordered ProTα and Neh2 to Keap1 occurs synergistically via preformed structural elements (PSEs) and coupled folding and binding, with a heavy bias towards PSEs, particularly for Neh2. Our results provide insights into the molecular mechanisms Neh2 and ProTα bind to Keap1, information that is useful for developing therapeutics to enhance the oxidative stress response.

Figure 1. Crystal structures of ProTα and Neh2 peptides bound to the Kelch domain of Keap1.

A) Cartoon B-Spline representations of the ProTα-Keap1 and Neh2-Keap1 crystal structures (PDB ids: 2Z32 and 1X2R respectively , . Residues Asn-41 to Glu-48 of ProTα and Leu-76 to Leu-84 of Neh2 (red) are shown bound to the Kelch domain of Keap1 (grey). B) Licorice representations of the i to i+3 residues of the β-turns from the crystal structures (⁴¹Asn-Glu-Glu-Asn⁴⁴ and ⁷⁷Asp-Glu-Glu-Thr⁸⁰, of ProTα and Neh2 respectively). C) Overlay of the ProTα (white) and Neh2 (grey) β-turns.

Figure 2. All-atom RMSD values between the MD and crystal structures.

The RMSD values were computed by subtracting the all-atom distance matrix at time t of the MD trajectories from the reference distance matrix determined from the crystal structures of the ProTα and Neh2 peptides bound to Keap1 (PDB ids: 2Z32 and 1X2R respectively) , . The distance matrices consisted of residues i through i+3 of the β-turn regions of the ProTα and Neh2 peptides determined from the crystal structures , .

Figure 3. Cαⁱ−Cαⁱ⁺³ distances and their deviations from their crystal structure distances.

Panels A and C show the C αⁱ−C αⁱ⁺³ distances. Panels B and D show the absolute deviations of C αⁱ−C αⁱ⁺³ distances from the corresponding distances in the crystal structures. Data were collected over the full 1.0 µs trajectories for the crystal structure peptides and the last 0.1 µs for the full-length ProTα and 32-mer Neh2. Deviations were calculated for C αⁱ−C αⁱ⁺³ pairs from the β-turns, determined from the crystal structures , , by subtraction of the i to i+3 distance at time t of the trajectory from the fixed distance of the corresponding atom pair from the crystal structures (PDB ids: 2Z32 and 1X2R) for ProTα and Neh2 respectively) , .

Figure 4. Overlay of the free and bound-state β-turns. Residues i through i+3 of the β-turns from the full-length ProT α and the 32-mer Neh2 MD structures were superimposed onto the corresponding residues from their bound state crystal structures.

Cluster centroids from the last 0.1 µs of the MD simulations (grey) were superimposed onto the corresponding C_α atoms from the crystal structures (pink) of ProTα and Neh2 bound to Keap1 (PDB ids: 2Z32 and 1X2R respectively) , . The single linkage clustering algorithm was used with a cutoff that included all structures from the last 0.1 µs. Hydrogens were added to the crystal structures for clarity. RMSD values were computed by subtracting the C_α, backbone or all-atom distance matrix of the centroid structures from the reference distance matrix determined from the crystal structures of the ProTα and Neh2 peptides bound to Keap1 (PDB ids: 2Z32 and 1X2R respectively) , .

Figure 5. Ramachandran plots for residues i to i+3 of the β-turns from the MD and crystal structures.

Red dots indicate the ϕ and ψ pair from the last 0.1 µs of the full-length ProTα and the 32-mer Neh2 trajectories. Blue circles indicate the angles of the starting structures. Green circles indicate the ϕ and ψ angle pair from the crystal structures (PDB ids: 2Z32 and 1X2R) , . Circular variance (C.V.) values and overlaid licorice representation snapshots from the last 0.1 µs of the simulations illustrate backbone mobility within the β-turns of ProTα and Neh2. Average circular variance values were calculated over the last 0.1 µs of the full-length ProTα and the 32-mer Neh2 peptide MD trajectories using the method described by MacArthur & Thornton .

Figure 6. Cα−Cα contacts in the MD structures.

A) Average C α−C α distances over the last 0.1 µs of the full-length ProTα and 32-mer Neh2 MD trajectories. Distances equal to or greater than 10 Å are colored dark red and distances equal to or less than 2 Å are colored dark blue. The C αⁱ−C αⁱ⁺³ atoms of the β-turns are indicated by the black boxes. B) Cartoon B-Spline representations colored by residue type of ther Keap1 binding regions of full-length ProTα and 32-mer Neh2 cluster centroids from the last 0.1 µs of the MD simulations. The single linkage clustering algorithm was used with a cutoff that included all structures from the last 0.1 µs. Residues comprising the XEEXGE Keap1-binding motifs are labeled. Directionality is indicated with the N and C labels.