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

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.

SUBMITTER: Parate S

PROVIDER: S-EPMC8194344 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundYinchen Wuling powder is often used to treat clinical hyperlipidemia, although its mechanism of action remains unclear. In this study, we aimed to investigate the active ingredients found in Yinchen Wuling powder and find its mechanism of action when treating hyperlipidemia, using a combination of network pharmacology, molecular docking, and molecular dynamics simulation approaches.MethodsThe TCMSP database was used to obtain the principle active ingredients found in Yinchen Wuling powder and the NCBI and DisGeNet databases were used to obtain the main target genes involved in hyperlipidemia, and the intersectional targets were obtained by EXCEL. We also used Cytoscape 3.7.2 software to construct a "Traditional Chinese Medicine-Active Ingredient-Target" network and use STRING platform to conduct "protein-protein interactional" (PPI) analyses on the intersection targets. Bioconductor software and RX 64 4.0.0 software were then used to perform GO functional enrichment analysis and KEGG pathway enrichment analysis on the targets. Molecular docking of core protein-ligand interactions was modeled using AutoDock Vina software. A simulation of molecular dynamics was conducted for the optimal core protein-ligand obtained by molecular docking using Amber18 software.ResultsA total of 63 active ingredients were found in Yinchen Wuling powder, corresponding to 175 targets, 508 hyperlipidemia targets, and 55 intersection targets in total. Cytoscape 3.7.2 showed that the key active ingredients were quercetin, isorhamnetin, taxifolin, demethoxycapillarisin, and artepillin A. The PPI network showed that the key proteins involved were AKT1, IL6, VEGFA, and PTGS2. GO enrichment analysis found that genes were enriched primarily in response to oxygen levels and nutrient levels of the vesicular lumen and were associated with membrane rafts. These were mainly enriched in AGE-RAGE (advanced glycation end products-receptor for advanced glycation end products) signaling pathway in diabetic complications, fluid shear stress, and atherosclerosis, as well as other pathways. The molecular docking results indicated key binding activity between PTGS2-quercetin, PTGS2-isorhamnetin, and PTGS2-taxifolin. Results from molecular dynamics simulations showed that PTGS2-quercetin, PTGS2-isorhamnetin, and PTGS2-taxifolin bound more stably, and their binding free energies were PTGS2-quercetin -29.5 kcal/mol, PTGS2-isorhamnetin -32 kcal/mol, and PTGS2-taxifolin -32.9 kcal/mol.ConclusionThis study is based on network pharmacology and reveals the potential molecular mechanisms involved in the treatment of hyperlipidemia by Yinchen Wuling powder.

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

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