Oncogenic mutations on Rac1 affect global intrinsic dynamics underlying GTP and PAK1 binding

Rac1 is a small member of the Rho GTPase family. One of the most important downstream effectors of Rac1 is a serine/threonine kinase, p21-activated kinase 1 (PAK1). Mutational activation of PAK1 by Rac1 has oncogenic signaling effects. Here, although we focus on Rac1-PAK1 interaction by atomic-force-microscopy-based single-molecule force spectroscopy experiments, we explore the effect of active mutations on the intrinsic dynamics and binding interactions of Rac1 by Gaussian network model analysis and molecular dynamics simulations. We observe that Rac1 oncogenic mutations are at the hinges of three global modes of motion, suggesting the mechanical changes as potential markers of oncogenicity. Indeed, the dissociation of wild-type Rac1-PAK1 complex shows two distinct unbinding dynamic states that are reduced to one with constitutively active Q61L and oncogenic Y72C mutant Rac1, as revealed by single-molecule force spectroscopy experiments. Q61L and Y72C mutations change the mechanics of the Rac1-PAK1 complex by increasing the elasticity of the protein and slowing down the transition to the unbound state. On the other hand, Rac1’s intrinsic dynamics reveal more flexible GTP and PAK1-binding residues on switches I and II with Q61L, Y72C, oncogenic P29S and Q61R, and negative T17N mutations. The cooperativity in the fluctuations of GTP-binding sites around the p-loop and switch I decreases in all mutants, mostly in Q61L, whereas some PAK1-binding residues display enhanced coupling with GTP-binding sites in Q61L and Y72C and within each other in P29S. The predicted binding free energies of the modeled Rac1-PAK1 complexes show that the change in the dynamic behavior likely means a more favorable PAK1 interaction. Overall, these findings suggest that the active mutations affect intrinsic functional dynamic events and alter the mechanics underlying the binding of Rac1 to GTP and upstream and downstream partners including PAK1

The structural network of Interleukin-10 and its implications in inflammation and cancer

Background: Inflammation has significant roles in all phases of tumor development, including initiation, progression and metastasis. Interleukin-10 (IL-10) is a well-known immuno-modulatory cytokine with an anti-inflammatory activity. Lack of IL-10 allows induction of pro-inflammatory cytokines and hinders anti-tumor immunity, thereby favoring tumor growth. The IL-10 network is among the most important paths linking cancer and inflammation. The simple node-and-edge network representation is useful, but limited, hampering the understanding of the mechanistic details of signaling pathways. Structural networks complete the missing parts, and provide details. The IL-10 structural network may shed light on the mechanisms through which disease-related mutations work and the pathogenesis of malignancies. Results: Using PRISM (a PRotein Interactions by Structural Matching tool), we constructed the structural network of IL-10, which includes its first and second degree protein neighbor interactions. We predicted the structures of complexes involved in these interactions, thereby enriching the available structural data. In order to reveal the significance of the interactions, we exploited mutations identified in cancer patients, mapping them onto key proteins of this network. We analyzed the effect of these mutations on the interactions, and demonstrated a relation between these and inflammation and cancer. Our results suggest that mutations that disrupt the interactions of IL-10 with its receptors (IL-10RA and IL-10RB) and alpha 2-macroglobulin (A2M) may enhance inflammation and modulate anti-tumor immunity. Likewise, mutations that weaken the A2M-APP (amyloid precursor protein) association may increase the proliferative effect of APP through preventing beta-amyloid degradation by the A2M receptor, and mutations that abolish the A2M-Kallikrein-13 (KLK13) interaction may lead to cell proliferation and metastasis through the destructive effect of KLK13 on the extracellular matrix. Conclusions: Prediction of protein-protein interactions through structural matching can enrich the available cellular pathways. In addition, the structural data of protein complexes suggest how oncogenic mutations influence the interactions and explain their potential impact on IL-10 signaling in cancer and inflammation

Fundamentals of Molecular Docking and Comparative Analysis of Protein–Small-Molecule Docking Approaches

Proteins (e.g., enzymes, receptors, hormones, antibodies, transporter proteins, etc.) seldom act alone in the cell, and their functions rely on their interactions with various partners such as small molecules, other proteins, and/or nucleic acids. Molecular docking is a computational method developed to model these interactions at the molecular level by predicting the 3D structures of complexes. Predicting the binding site and pose of a protein with its partner through docking can help us to unveil protein structure-function relationship and aid drug design in numerous ways. In this chapter, we focus on the fundamentals of protein docking by describing docking methods including search algorithm, scoring, and assessment steps as well as illustrating recent successful applications in drug discovery. We especially address protein–small-molecule (drug) docking by comparatively analyzing available tools implementing different approaches such as ab initio, structure-based, ligand-based (pharmacophore-/shape-based), information-driven, and machine learning approaches

The Structural Pathway of Interleukin 1 (IL-1) Initiated Signaling Reveals Mechanisms of Oncogenic Mutations and SNPs in Inflammation and Cancer

<div><p>Interleukin-1 (IL-1) is a large cytokine family closely related to innate immunity and inflammation. IL-1 proteins are key players in signaling pathways such as apoptosis, TLR, MAPK, NLR and NF-κB. The IL-1 pathway is also associated with cancer, and chronic inflammation increases the risk of tumor development via oncogenic mutations. Here we illustrate that the structures of interfaces between proteins in this pathway bearing the mutations may reveal how. Proteins are frequently regulated via their interactions, which can turn them ON or OFF. We show that oncogenic mutations are significantly at or adjoining interface regions, and can abolish (or enhance) the protein-protein interaction, making the protein constitutively active (or inactive, if it is a repressor). We combine known structures of protein-protein complexes and those that we have predicted for the IL-1 pathway, and integrate them with literature information. In the reconstructed pathway there are 104 interactions between proteins whose three dimensional structures are experimentally identified; only 15 have experimentally-determined structures of the interacting complexes. By predicting the protein-protein complexes throughout the pathway via the PRISM algorithm, the structural coverage increases from 15% to 71%. <i>In silico</i> mutagenesis and comparison of the predicted binding energies reveal the mechanisms of how oncogenic and single nucleotide polymorphism (SNP) mutations can abrogate the interactions or increase the binding affinity of the mutant to the native partner. Computational mapping of mutations on the interface of the predicted complexes may constitute a powerful strategy to explain the mechanisms of activation/inhibition. It can also help explain how an oncogenic mutation or SNP works.</p></div

t-test for COSMIC mutations mapped onto the interface region including the nearby residues.

<p>t-test for COSMIC mutations mapped onto the interface region including the nearby residues.</p

The distribution of oncogenic mutations and SNPs on structures of interacting protein-protein complexes in IL-1 pathway.

<p>The distribution of oncogenic mutations and SNPs on structures of interacting protein-protein complexes in IL-1 pathway.</p

IL-1 signaling pathway reconstructed by combining related pathways and information from the literature.

<p> This detailed map of IL-1 signaling presents the protein-protein interactions and the resulting cellular events. The colored nodes represent proteins having experimentally identified 3D structures and the white nodes are the proteins without 3D structures. The edges represent protein-protein interactions (straight/dashed arrows relate to available/unavailable 3D structures of proteins) or associations leading to cellular events such as cell cycle or gene expression (dashed arrows beginning with circular heads).</p

The effect of mutations on the MKK4-JNK3, MKK7-JNK3 and IL1A(IL-1α)-IL1R1 interactions.

<p>The wild type target structures are energetically minimized, related residues are mutated and PRISM is re-run to predict the protein-protein interactions.</p>*<p>The new reference interactions for comparisons are the predicted interactions between energetically minimized wild type targets and for the three cases they are: MKK4-JNK3 interaction predicted using 1p4oAB template with a predicted binding energy of −12.66 energy units; MKK7-JNK3 predicted using 1ftaAC template with a predicted binding energy of −11.66 energy units; and IL1A-IL1R1 predicted using 1itbAB template with a predicted binding energy of −19.5 energy units.</p

The PRISM algorithm flow.

<p>Two sets are given as the input: template and target. Four consecutive steps are executed to produce the output set which is composed of the structures of protein-protein complexes predicted to have the lowest binding energies. In this figure, the template set contains only one member for visualization simplicity, but it is important to note that the default template set of the algorithm is composed of 7922 interface members.</p

Structures of protein-protein complexes mapped to the IL-1 signaling pathway.

<p><b>A.</b> Overall distribution of experimental and predicted complexes on the pathway. Dark blue interactions represent the experimentally determined complex structures in the PDB, red interactions represent the predicted complexes with predicted binding energies lower than −10 energy units and yellow interactions represent the interactions for which neither experimental nor computational data exists. <b>B.</b> Predicted structures (Template PDB code+Target PDB codes+Energy value): IL1α-IL1R1 (1itbAB 2l5x 4depB −71.92); IL1α-IL1RAP (1itbAB 2l5x 4depC −49.25); IL1R1-MYD88 (1gylAB IL1R1 (model) 2z5v −23.8); IL1R1-TOLLIP (1oh0AB IL1R1 (model) 1wgl −18.85); IL1RAP-MYD88 (1p65AB IL1RAP (model) 2z5v −31.72); MYD88-TOLLIP (1yrlAC 3mopA 1wgl −11.04); MYD88-TRAF6 (1vjlAB 2js7 1lb6 −37.01); TRAF6-IRF7 (1g8<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003470#pcbi-1003470-t002" target="_blank">tAB 2o</a>61 3hct −25.83). The blue color represents the proteins that precede its partners in the information flow.</p