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. 2021 May 28:8:655035.
doi: 10.3389/fmolb.2021.655035. eCollection 2021.

Exploring the Binding Interaction of Raf Kinase Inhibitory Protein With the N-Terminal of C-Raf Through Molecular Docking and Molecular Dynamics Simulation

Shraddha Parate  1 , Shailima Rampogu  2 , Gihwan Lee  1 , Jong Chan Hong  1 , Keun Woo Lee  2
Affiliations

Exploring the Binding Interaction of Raf Kinase Inhibitory Protein With the N-Terminal of C-Raf Through Molecular Docking and Molecular Dynamics Simulation

Shraddha Parate et al. Front Mol Biosci. .

Abstract

Protein-protein interactions are indispensable physiological processes regulating several biological functions. Despite the availability of structural information on protein-protein complexes, deciphering their complex topology remains an outstanding challenge. Raf kinase inhibitory protein (RKIP) has gained substantial attention as a favorable molecular target for numerous pathologies including cancer and Alzheimer's disease. RKIP interferes with the RAF/MEK/ERK signaling cascade by endogenously binding with C-Raf (Raf-1 kinase) and preventing its activation. In the current investigation, the binding of RKIP with C-Raf was explored by knowledge-based protein-protein docking web-servers including HADDOCK and ZDOCK and a consensus binding mode of C-Raf/RKIP structural complex was obtained. Molecular dynamics (MD) simulations were further performed in an explicit solvent to sample the conformations for when RKIP binds to C-Raf. Some of the conserved interface residues were mutated to alanine, phenylalanine and leucine and the impact of mutations was estimated by additional MD simulations and MM/PBSA analysis for the wild-type (WT) and constructed mutant complexes. Substantial decrease in binding free energy was observed for the mutant complexes as compared to the binding free energy of WT C-Raf/RKIP structural complex. Furthermore, a considerable increase in average backbone root mean square deviation and fluctuation was perceived for the mutant complexes. Moreover, per-residue energy contribution analysis of the equilibrated simulation trajectory by HawkDock and ANCHOR web-servers was conducted to characterize the key residues for the complex formation. One residue each from C-Raf (Arg398) and RKIP (Lys80) were identified as the druggable "hot spots" constituting the core of the binding interface and corroborated by additional long-time scale (300 ns) MD simulation of Arg398Ala mutant complex. A notable conformational change in Arg398Ala mutant occurred near the mutation site as compared to the equilibrated C-Raf/RKIP native state conformation and an essential hydrogen bonding interaction was lost. The thirteen binding sites assimilated from the overall analysis were mapped onto the complex as surface and divided into active and allosteric binding sites, depending on their location at the interface. The acquired information on the predicted 3D structural complex and the detected sites aid as promising targets in designing novel inhibitors to block the C-Raf/RKIP interaction.

Keywords: C-Raf; HADDOCK; MM/PBSA; RKIP; ZDOCK; binding sites prediction; molecular dynamics simulation; protein-protein docking.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
(A) Structural alignment and (B) comparative backbone root mean square deviation (RMSD) analysis of docked structural complexes generated from HADDOCK (mint green) and ZDOCK (orange) web-servers.
FIGURE 2
FIGURE 2
Comparative backbone root mean square deviation (RMSD) analysis of wild-type (WT) structural complex and constructed mutant complexes.
FIGURE 3
FIGURE 3
Comparative backbone root mean square fluctuation (RMSF) analysis of wild-type (WT) structural complex and constructed mutant complexes for residues of (A) C-Raf and (B) RKIP.
FIGURE 4
FIGURE 4
Binding mode of C-Raf/RKIP structural complex and MM/GBSA analysis. (A) C-Raf (purple) and RKIP (mint green) shown in surface representation. (B)Close-up view of the intermolecular interactions between C-Raf and RKIP. Interacting residues are represented in stick form. Hydrogen, hydrophobic and electrostatic bonds are shown as green, pink and brown dashed lines, respectively. Per-residue energy contribution (kcal/mol) of key residues in (C) C-Raf and (D) RKIP.
FIGURE 5
FIGURE 5
Comparison of the conformational change at the Arg398Ala mutation site as compared with the wild-type (WT) C-Raf/RKIP structural complex obtained through molecular dynamics (MD) simulations.(A)The equilibrated native state conformation of WT (mint green) superimposed with the Arg398Ala (mauve) representative snapshot extracted at 180°ns. (B) Enlarged view of the conformational change at the Arg398Ala mutation site. Conventional hydrogen bonds are displayed as dark green dashed lines, while carbon-hydrogen bond is shown as a light green dashed line.
FIGURE 6
FIGURE 6
Potential binding sites in the C-Raf/RKIP complex presented as surface with different colors in the structure.
See this image and copyright information in PMC

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