Abstract
The death-inducing signaling complex (DISC) is a fundamental multiprotein complex, which triggers the extrinsic apoptosis pathway through stimulation by death ligands. DISC consists of different death domain (DD) and death effector domain (DED) containing proteins such as the death receptor Fas (CD95) in complex with FADD, procaspase-8, and cFLIP. Despite many experimental and theoretical studies in this area, there is no global agreement neither on the DISC architecture nor on the mechanism of action of the involved species. In the current work, we have tried to reconstruct the DISC structure by identifying key protein interactions using a new protein–protein docking meta-approach. We combined the benefits of five of the most employed protein–protein docking engines, HADDOCK, ClusPro, HDOCK, GRAMM-X, and ZDOCK, in order to improve the accuracy of the predicted docking complexes. Free energy of binding and hot spot interacting residues were calculated and determined for each protein–protein interaction using molecular mechanics generalized Born surface area and alanine scanning techniques, respectively. In addition, a series of in-cellulo protein-fragment complementation assays were conducted to validate the protein–protein docking procedure. The results show that the DISC formation initiates by dimerization of adjacent FasDD trimers followed by recruitment of FADD through homotypic DD interactions with the oligomerized death receptor. Furthermore, the in-silico outcomes indicate that cFLIP cannot bind directly to FADD; instead, cFLIP recruitment to the DISC is a hierarchical and cooperative process where FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with cFLIP. Finally, a possible structure of the entire DISC is proposed based on the docking results.
Highlights
Proteins play a principal role in many essential biological processes within the living organisms, ranging from signal transduction and enzyme catalysis to gene expression and metabolism
The Fas opening has two consequences: first it discloses a hydrophobic patch which serves as the binding site of FADDDD (Figure 4b and c), and second it allows homodimerization of two open Fas molecules through interactions between their stem helices in a Fas−Fas bridge conformation (Figure 4c).[44]
Using a meta-approach for protein−protein docking in which we combined the data obtained from the protein−protein docking engines HADDOCK, ClusPro, HDOCK, GRAMM-X, and ZDOCK, the structures of the different dimeric components of the death-inducing signaling complex (DISC) complex were predicted to high accuracy
Summary
Proteins play a principal role in many essential biological processes within the living organisms, ranging from signal transduction and enzyme catalysis to gene expression and metabolism. Proteins rarely perform their in vivo tasks as isolated species; instead, they interact with other proteins and other biomolecules such as RNA and DNA in sophisticated “molecular networks”. It has been demonstrated that more than 80% of all proteins are involved in at least one protein−protein interaction (PPI).[1] It is estimated that there are 600 000 different PPIs in the human interactome[2,3] which exceeds the number of proteins in the proteome by one order of magnitude.[4] PPIs are as important as the proteins themselves for cell survival.[5] a profound understanding of PPIs and identifying the related key interacting residues is necessary in order to design drug molecules which can interfere with specific pathways as novel therapeutic disease intervention.[6]
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