Contribution of Mass Spectrometry-Based Proteomics to the Understanding of TNF‑α Signaling

NF-κB is a family of ubiquitous dimeric transcription factors that play a role in a myriad of cellular processes, ranging from differentiation to stress response and immunity. In inflammation, activation of NF-κB is mediated by pro-inflammatory cytokines, in particular the prototypic cytokines IL-1β and TNF-α, which trigger the activation of complex signaling cascades. In spite of decades of research, the system level understanding of TNF-α signaling is still incomplete. This is partially due to the limited knowledge at the proteome level. The objective of this review is to summarize and critically evaluate the current status of the proteomic research on TNF-α signaling. We will discuss the merits and flaws of the existing studies as well as the insights that they have generated into the proteomic landscape and architecture connected to this signaling pathway. Besides delineating past and current trends in TNF-α proteomic research, we will identify research directions and new methodologies that can further contribute to characterize the TNF-α associated proteome in space and time

Cross-Link Guided Molecular Modeling with ROSETTA

<div><p>Chemical cross-links identified by mass spectrometry generate distance restraints that reveal low-resolution structural information on proteins and protein complexes. The technology to reliably generate such data has become mature and robust enough to shift the focus to the question of how these distance restraints can be best integrated into molecular modeling calculations. Here, we introduce three workflows for incorporating distance restraints generated by chemical cross-linking and mass spectrometry into <i>ROSETTA</i> protocols for comparative and <i>de novo</i> modeling and protein-protein docking. We demonstrate that the cross-link validation and visualization software <i>Xwalk</i> facilitates successful cross-link data integration. Besides the protocols we introduce <i>XLdb</i>, a database of chemical cross-links from 14 different publications with 506 intra-protein and 62 inter-protein cross-links, where each cross-link can be mapped on an experimental structure from the Protein Data Bank. Finally, we demonstrate on a protein-protein docking reference data set the impact of virtual cross-links on protein docking calculations and show that an inter-protein cross-link can reduce on average the RMSD of a docking prediction by 5.0 Å. The methods and results presented here provide guidelines for the effective integration of chemical cross-link data in molecular modeling calculations and should advance the structural analysis of particularly large and transient protein complexes via hybrid structural biology methods.</p></div

Overview of 15 proteins from the PP2A network for which comparative models were generated.

<p>Listed next to the protein UniProt entry names are information on the template PDB structure, the sequence identity between the target and template protein sequences, the number of experimental cross-links collected for each protein, the largest number of cross-links that were satisfied by each protein’s best model, the minimum RMSD value to the template PDB structure observed during entire simulation, the RMSD value of the model that satisfied most cross-links while having the lowest RMSD value, the score and the rank position of that model.</p

Comparative modeling calculations and chemical cross-link data validation on 15 proteins from the PP2A interaction network.

<p>(A) ROSETTA energy score versus RMSD plots for all proteins. Template structures (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073411#pone-0073411-t001" target="_blank">Table 1</a>) served as a reference for the RMSD calculations. Green colored dots highlight those models that satisfy most chemical cross-links; their numbers are indicated at the top right corner of each scatter plot. (B) For each protein from (A), only the model with the largest RMSD value is plotted demonstrating the prediction improvement with the increasing number of chemical cross-links.</p

Computational workflows for cross-link guided molecular modeling centered on <i>ROSETTA</i> protocols and <i>Xwalk</i> software.

<p>(A) Comparative modeling. (B) <i>De novo</i> modeling with partial structural information. (C) Protein-protein docking. Flowcharts were generated using <a href="https://www.draw.io" target="_blank">https://www.draw.io</a>.</p

Prediction of the IgBP1-PP2AA protein topology using 7 inter-protein cross-links, 11 intra-protein cross-links and 10 mono-links.

<p>(A) Structural model of the lowest scoring models from the 4 largest clusters, showing the PP2AA protein in purple color and the IgBP1 protein in dark green color. The solid cartoon representation corresponds to the cluster representative of the largest cluster, while the transparent IgBP1 models are cluster representatives of the 2nd, 3rd and 4th largest cluster. Intra-links with their shortest SAS distance path are shown as green colored chains of spheres, inter-links are shown in red and mono-links are highlighted as blue spheres. In addition, black spheres indicate previously mutated amino acids that were shown to be involved in forming the interface of IgBP1 and PP2AA. (B) Overview of the <i>ROSETTA</i> energy scores for all models that satisfied at least 6 inter-protein cross-links by means of the Euclidean distance measure are shown as empty grey circles. The RMSD was calculated to the cluster representative of the largest cluster. Models satisfying at least 6 inter-protein cross-links by means of the SAS distance measure and having a binding interface size ≥900 Ų are highlighted in blue, while the cluster representatives of the 4 largest clusters are highlighted as red colored circles.</p

A selected list of 16 binary protein complexes from the “difficult” and “medium difficult” category of the protein docking benchmark dataset version 4.0 [39].

<p>Each complex has more than 7 predicted (virtual) inter-protein cross-links and was employed to test the impact of cross-links on protein docking calculations. Best models correspond to the models with the shortest mean SAS distance for all 7 cross-links. L-RMSD corresponds to the RMSD value among the smaller protein partners also known as ligands.</p

Chemical cross-links on the regulatory subunit 2ABG of PP2A might have originated from a stable intermediate folding state.

<p>(A) The comparative model that is most similar to its template structure 2ABA satisfies only 6 of 18 intra-protein cross-links. (B) In contrast, the comparative model that satisfies with 13 cross-links most of the cross-link data has a RMSD of 19.5 Å and is partially unfolded. Green chain of spheres indicate the shortest path between cross-linked lysine pairs that have an SAS distance ≤34.0 Å.</p

Overview of 6 proteins used as a reference data set for the comparative modeling workflow.

<p>For more information regarding the columns, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073411#pone-0073411-t001" target="_blank">Table 1</a>.</p><p><b>Note, that the RMSD values were calculated between the models and the native structure and not the template structure as in </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073411#pone-0073411-t001" target="_blank"><b>Table 1</b></a><b>.</b></p

Localization of the C-terminal domain of IgBP1 with chemical cross-link data.

<p>(A) <b><i>ROSETTA</i></b> energy score versus RMSD plot for full-length models of IgBP1, with one of the best models acting as a reference structure for the RMSD calculation. Only models below an energy score of 650 are shown. Grey empty circles are models that satisfy more than 60 cross-links by Euclidean distance measure. Blue circles depict models that satisfy more than 60 cross-links by means of the SAS distance measure. The five red circles have been chosen as best models with RMSD ≤10.0 Å to the N-terminal template structure of mouse IgBP1 (PDB-ID: 3QC1). (B) Structure of the five best models. The structures are colored from blue to red between the N and C-terminus. The models were superimposed on their N-terminal domain highlighting the co-location of their C-terminal domain.</p