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High-resolution structural characterization of Noxa, an intrinsically disordered protein, by microsecond molecular dynamics simulations - PubMed

High-resolution structural characterization of Noxa, an intrinsically disordered protein, by microsecond molecular dynamics simulations

L Michel Espinoza-Fonseca et al. Mol Biosyst. 2015 Jul.

Abstract

High-resolution characterization of the structure and dynamics of intrinsically disordered proteins (IDPs) remains a challenging task. Consequently, a detailed understanding of the structural and functional features of IDPs remains limited, as very few full-length disordered proteins have been structurally characterized. We have performed microsecond-long molecular dynamics (MD) simulations of Noxa, the smallest member of the large Bcl-2 family of apoptosis regulating proteins, to characterize in atomic-level detail the structural features of a disordered protein. A 2.5 μs MD simulation starting from an unfolded state of the protein revealed the formation of a central antiparallel β-sheet structure flanked by two disordered segments at the N- and C-terminal ends. This topology is in reasonable agreement with protein disorder predictions and available experimental data. We show that this fold plays an essential role in the intracellular function and regulation of Noxa. We demonstrate that unbiased MD simulations in combination with a modern force field reveal structural and functional features of disordered proteins at atomic-level resolution.

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Figures

Fig. 1
Fig. 1. Disorder analysis of Noxa using multiple prediction algorithms

The plot shows the disorder propensity of each amino acid in Noxa. All prediction algorithms use the same disorder scale: residues with values between 0 and 0.5 are considered structurally ordered, whereas residues with disorder propensity values between 0.5 and 1 are considered structurally disordered. The sequence of Noxa is shown at the top of the graph, and residues that are likely to be disordered based on disorder prediction algorithms are shown in red.

Fig. 2
Fig. 2. Time-dependent structural fluctuations of Noxa

(A) Evolution of the secondary structure of Noxa in the 2.5-μs trajectory. Secondary structure is colored as α-helix (pink), 310-helix (blue), β-strand (yellow) turn (cyan), and coil (white). (B) Snapshots illustrating the key structural features of Noxa as determined by 5-D dPCA clustering analysis. Each cluster is indicated in Roman numerals; the location of each cluster in the trajectory is shown with keys above panel A. The structure of Noxa is represented in ribbons and colored based on secondary structure, and N- and C-termini are shown as blue and red spheres, respectively.

Fig. 3
Fig. 3. dPCA-based free energy landscapes of Noxa

Free energy landscapes were obtained using combinations of principal components 1–2, 2–3, 3–4 and 4–5 as reaction coordinates. In all cases, the free energy landscapes features multiple minima separated by relatively small energy barriers.

Fig. 4
Fig. 4. RMSF values calculated for Noxa

RMSF values were calculated between 1–2.5 μs by aligning the Cα atoms of Noxa onto residues A19-V23 and L36-N40. A cartoon representation of the secondary structure content of of Noxa is shown at the top of the plot. The shaded area in the plot indicates the location of the BH3 motif.

Fig. 5
Fig. 5. Time-dependent changes in the distance distances E22-R39 and D20-K41

We calculated the distances between Cδ of glutamate and Cζ and Nζ of arginine and lysine, respectively. The formations of salt bridges at different times are shown as grey cartoons; the residues involved in salt-bridge formation and β-nucleation are shown as sticks. The main plot shows the distances calculated only for the first 1 μs in the trajectory; the inset plot shows the changes in interresidue distances for the entire 2.5 μs of simulation time.

Fig. 6
Fig. 6. Occurrence of interstrand hydrogen bonds

% H-bond was calculated between t=1 μs and t=2.5 μs using individual structures at time intervals of 0.04 μs. The cartoon shows the location of the hydrogen bond donors and acceptors located at the interface of the β-sheet. Backbone atoms are shown as van der Waals spheres and colored according to atom type: oxygen (red), nitrogen (blue), carbon (cyan) and hydrogen (white).

Fig. 7
Fig. 7. Claw-like structure of the BH3 motif

The structure of Noxa corresponds to the representative cluster V shown in Fig. 3C. Noxa is shown as cyan ribbons and the side chain residues of the BH3 motif (residues L29-K35) are shown as sticks.

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