Binding of the bacteriophage P22 N-peptide to the boxB RNA motif studied by molecular dynamics simulations

Abstract

Protein-RNA interactions are important for many cellular processes. The Nut-utilization site (N)-protein of bacteriophages contains an N-terminal arginine-rich motif that undergoes a folding transition upon binding to the boxB RNA hairpin loop target structure. Molecular dynamics simulations were used to investigate the dynamics of the P22 N-peptide-boxB complex and to elucidate the energetic contributions to binding. In addition, the free-energy changes of RNA and peptide conformational adaptation to the bound forms, as well as the role of strongly bound water molecules at the peptide-RNA interface, were studied. The influence of peptide amino acid substitutions and the salt dependence of interaction were investigated and showed good agreement with available experimental results. Several tightly bound water molecules were found at the RNA-binding interface in both the presence and absence of N-peptide. Explicit consideration of the waters resulted in shifts of calculated contributions during the energetic analysis, but overall similar binding energy contributions were found. Of interest, it was found that the electrostatic field of the RNA has a favorable influence on the coil-to-alpha-helix transition of the N-peptide already outside of the peptide-binding site. This result may have important implications for understanding peptide-RNA complex formation, which often involves coupled folding and association processes. It indicates that electrostatic interactions near RNA molecules can lead to a shift in the equilibrium toward the bound form of an interacting partner before it enters the binding pocket.

Figure 1

RMSD (heavy atoms) of sampled conformations of complex, peptide, and RNA from the native structure (Protein Data Bank: 1A4T) versus simulation time. (A) RMSD of the N-peptide-boxB RNA complex (black), RNA in complex (red), and peptide in complex (green/light gray) starting from the experimental structure. (B) RMSD of isolated RNA (red/dark gray) and isolated peptide (green/light gray) starting from the experimental structure.

Figure 2

Superposition (in stereo) of the final structures (red/light gray) of complex (A) and isolated RNA hairpin (B) on the experimental starting structures (blue/dark gray). The RNA hairpin is shown in stick representation and the N-peptide is in backbone tube representation. (C) Stereo view of the N-peptide (tube representation) taken from the final stage of simulation of the complex (blue/dark gray) and the final structure of the MD simulation of the isolated peptide (red/light gray).

Figure 3

Stabilization of the coil-to-helix transition near the RNA binding site. (Upper panel) Schematic illustration of the process. (Lower panel) MM/PBSA free-energy contributions averaged over the last 10 ns of the MD simulations (2500 snapshots). Structures representing the helical bound form were taken from the simulation of the complex (helix shifted by 15 Å from the binding site). The structures of the isolated N-peptide were taken from the simulation of the isolated peptide shifted to the same position 15 Å from the binding site. The placement was sufficiently far from the RNA to avoid any sterical overlap.

Figure 4

Change in the calculated binding free energy upon substitution by alanine. Negative ΔΔG values correspond to unfavorable substitutions, and positive values indicate improved binding due to alanine mutation. The change in the solvent-accessible surface area that is buried at the protein-RNA interface for each amino acid residues is also indicated. Results are given for the PB (A) and GB (B) approaches.