Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Crystal Structure Reveals the Full Ras-Raf Interface and Advances Mechanistic Understanding of Raf Activation.

ABSTRACT: Ras and Raf-kinase interact through the Ras-binding (RBD) and cysteine-rich domains (CRD) of Raf to signal through the mitogen-activated protein kinase pathway, yet the molecular mechanism leading to Raf activation has remained elusive. We present the 2.8 Å crystal structure of the HRas-CRaf-RBD_CRD complex showing the Ras-Raf interface as a continuous surface on Ras, as seen in the KRas-CRaf-RBD_CRD structure. In molecular dynamics simulations of a Ras dimer model formed through the α4-α5 interface, the CRD is dynamic and located between the two Ras protomers, poised for direct or allosteric modulation of functionally relevant regions of Ras and Raf. We propose a molecular model in which Ras binding is involved in the release of Raf autoinhibition while the Ras-Raf complex dimerizes to promote a platform for signal amplification, with Raf-CRD centrally located to impact regulation and function.

SUBMITTER: Cookis T

PROVIDER: S-EPMC8301913 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Crystal Structure Reveals the Full Ras-Raf Interface and Advances Mechanistic Understanding of Raf Activation.

Cookis Trinity T Mattos Carla C

Biomolecules 20210707 7

Ras and Raf-kinase interact through the Ras-binding (RBD) and cysteine-rich domains (CRD) of Raf to signal through the mitogen-activated protein kinase pathway, yet the molecular mechanism leading to Raf activation has remained elusive. We present the 2.8 Å crystal structure of the HRas-CRaf-RBD_CRD complex showing the Ras-Raf interface as a continuous surface on Ras, as seen in the KRas-CRaf-RBD_CRD structure. In molecular dynamics simulations of a Ras dimer model formed through the α4-α5 inter ...[more]

PMID: 34356620

Similar Datasets

Ras-Mediated Activation of the Raf Family Kinases.

Project description:The extracellular signal-regulated kinase (ERK) cascade comprised of the Raf, MEK, and ERK protein kinases constitutes a key effector cascade used by the Ras GTPases to relay signals regulating cell growth, survival, proliferation, and differentiation. Of the ERK cascade components, the regulation of the Raf kinases is by far the most complex, involving changes in subcellular localization, protein and lipid interactions, as well as alterations in the Raf phosphorylation state. The Raf kinases interact directly with active, membrane-localized Ras, and this interaction is often the first step in the Raf activation process, which ultimately results in ERK activation and the downstream phosphorylation of cellular targets that will specify a particular biological response. Here, we will examine our current understanding of how Ras promotes Raf activation, focusing on the molecular mechanisms that contribute to the Raf activation/inactivation cycle.

| S-EPMC6311149 | biostudies-literature

Caenorhabditis elegans CNK-1 promotes Raf activation but is not essential for Ras/Raf signaling.

Project description:Connector enhancer of Ksr (CNK) is a conserved multidomain protein essential for Ras signaling in Drosophila melanogaster and thought to be involved in Raf kinase activation. However, the precise role of CNK in Ras signaling is not known, and mammalian CNKs are proposed to have distinct functions. Caenorhabditis elegans has a single CNK homologue, cnk-1. Here, we describe the role of cnk-1 in C. elegans Ras signaling and its requirements for LIN-45 Raf activation. We find that cnk-1 positively regulates multiple Ras signaling events during development, but, unlike Drosophila CNK, cnk-1 does not appear to be essential for signaling. cnk-1 mutants appear to be normal but show cell-type-specific genetic interactions with mutations in two other Ras pathway scaffolds/adaptors ksr-1 and sur-8. Genetic epistasis using various activated LIN-45 Raf transgenes shows that CNK-1 promotes LIN-45 Raf activation at a step between the dephosphorylation of inhibitory sites in the regulatory domain and activating phosphorylation in the kinase domain. Our data are consistent with a model in which CNK promotes Raf phosphorylation/activation through membrane localization, oligomerization, or association with an activating kinase.

| S-EPMC1187957 | biostudies-literature

Crystal Structure of the full-length Japanese encephalitis virus NS5 reveals a conserved methyltransferase-polymerase interface.

Project description:The flavivirus NS5 harbors a methyltransferase (MTase) in its N-terminal ≈ 265 residues and an RNA-dependent RNA polymerase (RdRP) within the C-terminal part. One of the major interests and challenges in NS5 is to understand the interplay between RdRP and MTase as a unique natural fusion protein in viral genome replication and cap formation. Here, we report the first crystal structure of the full-length flavivirus NS5 from Japanese encephalitis virus. The structure completes the vision for polymerase motifs F and G, and depicts defined intra-molecular interactions between RdRP and MTase. Key hydrophobic residues in the RdRP-MTase interface are highly conserved in flaviviruses, indicating the biological relevance of the observed conformation. Our work paves the way for further dissection of the inter-regulations of the essential enzymatic activities of NS5 and exploration of possible other conformations of NS5 under different circumstances.

| S-EPMC3738499 | biostudies-literature

Mechanistic Understanding of Lung Inflammation: Recent Advances and Emerging Techniques.

Project description:Acute respiratory distress syndrome (ARDS) is a life-threatening lung injury characterized by an acute inflammatory response in the lung parenchyma. Hence, it is considered as the most appropriate clinical syndrome to study pathogenic mechanisms of lung inflammation. ARDS is associated with increased morbidity and mortality in the intensive care unit (ICU), while no effective pharmacological treatment exists. It is very important therefore to fully characterize the underlying pathobiology and the related mechanisms, in order to develop novel therapeutic approaches. In vivo and in vitro models are important pre-clinical tools in biological and medical research in the mechanistic and pathological understanding of the majority of diseases. In this review, we will present data from selected experimental models of lung injury/acute lung inflammation, which have been based on clinical disorders that can lead to the development of ARDS and related inflammatory lung processes in humans, including ventilation-induced lung injury (VILI), sepsis, ischemia/reperfusion, smoke, acid aspiration, radiation, transfusion-related acute lung injury (TRALI), influenza, Streptococcus (S.) pneumoniae and coronaviruses infection. Data from the corresponding clinical conditions will also be presented. The mechanisms related to lung inflammation that will be covered are oxidative stress, neutrophil extracellular traps, mitogen-activated protein kinase (MAPK) pathways, surfactant, and water and ion channels. Finally, we will present a brief overview of emerging techniques in the field of omics research that have been applied to ARDS research, encompassing genomics, transcriptomics, proteomics, and metabolomics, which may recognize factors to help stratify ICU patients at risk, predict their prognosis, and possibly, serve as more specific therapeutic targets.

| S-EPMC9207257 | biostudies-literature

RAF inhibitors promote RAS-RAF interaction by allosterically disrupting RAF autoinhibition.

Project description:First-generation RAF inhibitors paradoxically induce ERK signaling in normal and tumor cells exhibiting RAS activity. Compound-induced RAF dimerization through stabilization of the RAF ON/active state by inhibitors has emerged as a critical contributing factor. RAF inhibitors also enhance RAS-RAF association. Although this event is thought to play a key role in priming RAF activation, the underlying mechanism is not known. Here we report that RAF inhibitors induce the disruption of intramolecular interactions between the kinase domain and its N-terminal regulatory region independently of RAS activity. This provides a molecular basis to explain the induction of RAS-RAF association by RAF inhibitors, as well as the co-operativity observed between RAS activity and RAF kinase inhibitors in driving RAF activation. Profiling of second-generation RAF inhibitors confirmed their improved mode of action, but also revealed liabilities that allowed us to discern two properties of an ideal RAF inhibitor: high-binding affinity to all RAF paralogs and maintenance of the OFF/autoinhibited state of the enzyme.

| S-EPMC5662619 | biostudies-literature

Frequent functional activation of RAS signalling not explained by RAS/RAF mutations in relapsed/refractory multiple myeloma.

Project description:RAS mutations are frequent in relapsed/refractory multiple myeloma (RRMM) but functional study in primary samples is scanty. Herein, in primary myeloma plasma cells of 17 suspected RRMM, functional activation of RAS signalling was studied by Western blot of phosphorylated ERK1/2 (phospho-ERK1/2). Moreover, activating mutations in KRAS, NRAS, BRAF, and ALK were studied by PCR and bidirectional direct sequencing. Furthermore, methylation of negative RAS signalling regulator genes, RASSF1A and RASD1, were analyzed by methylation-specific PCR. As evidenced by phospho-ERK1/2 over-expression, functional RAS activation was detected in 12 (75.0%) RRMM. Of patients with functional RAS activation, sequencing data showed only seven (58.3%) patients with one each had NRAS Q61H, NRAS Q61K, KRAS G12D, KRAS G12V, KRAS G13D, KRAS Q61P, or BRAF V600E mutation, whereas five (41.7%) patients had no RAS/RAF mutation. Conversely, patients without functional RAS activation had no RAS/RAF mutation. Moreover, none of the patients with functional RAS activation had ALK mutations, or methylation of RASSF1A and RASD1. Collectively, functional activation of RAS signalling was present in majority of RRMM but only about half (58.3%) accountable by RAS/RAF mutations. If verified in larger studies, clinical investigations of MEK inhibitors are warranted regardless of RAS/RAF mutations.

| S-EPMC6131153 | biostudies-literature

Advances in the Mechanistic Understanding of Iron Oxide Nanoparticles' Radiosensitizing Properties.

Project description:Among the plethora of nanosystems used in the field of theranostics, iron oxide nanoparticles (IONPs) occupy a central place because of their biocompatibility and magnetic properties. In this study, we highlight the radiosensitizing effect of two IONPs formulations (namely 7 nm carboxylated IONPs and PEG5000-IONPs) on A549 lung carcinoma cells when exposed to 225 kV X-rays after 6 h, 24 h and 48 h incubation. The hypothesis that nanoparticles exhibit their radiosensitizing effect by weakening cells through the inhibition of detoxification enzymes was evidenced by thioredoxin reductase activity monitoring. In particular, a good correlation between the amplification effect at 2 Gy and the residual activity of thioredoxin reductase was observed, which is consistent with previous observations made for gold nanoparticles (NPs). This emphasizes that NP-induced radiosensitization does not result solely from physical phenomena but also results from biological events.

| S-EPMC9823929 | biostudies-literature

Crystal Packing Reveals a Potential Autoinhibited KRAS Dimer Interface and a Strategy for Small-Molecule Inhibition of RAS Signaling.

Project description:KRAS GTPases harbor oncogenic mutations in more than 25% of human tumors. KRAS is considered to be largely undruggable due to the lack of a suitable small-molecule binding site. Here, we report a unique crystal structure of His-tagged KRASG12D that reveals a remarkable conformational change. The Switch I loop of one His-KRASG12D structure extends into the Switch I/II pocket of another His-KRASG12D in an adjacent unit cell to create an elaborate interface that is reminiscent of high-affinity protein-protein complexes. We explore the contributions of amino acids at this interface using alanine-scanning studies with alchemical free energy perturbation calculations based on explicit-solvent molecular dynamics simulations. Several interface amino acids were found to be hot spots as they contributed more than 1.5 kcal/mol to the protein-protein interaction. Computational analysis of the complex revealed the presence of two large binding pockets that possess physicochemical features typically found in pockets considered druggable. Small-molecule binding to these pockets may stabilize this autoinhibited structure of KRAS if it exists in cells to provide a new strategy to inhibit RAS signaling.

| S-EPMC10904212 | biostudies-literature

KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation.

Project description:The first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

| S-EPMC7895934 | biostudies-literature

Reverse gyrase--recent advances and current mechanistic understanding of positive DNA supercoiling.

Project description:Reverse gyrases are topoisomerases that introduce positive supercoils into DNA in an ATP-dependent reaction. They consist of a helicase domain and a topoisomerase domain that closely cooperate in catalysis. The mechanism of the functional cooperation of these domains has remained elusive. Recent studies have shown that the helicase domain is a nucleotide-regulated conformational switch that alternates between an open conformation with a low affinity for double-stranded DNA, and a closed state with a high double-stranded DNA affinity. The conformational cycle leads to transient separation of DNA duplexes by the helicase domain. Reverse gyrase-specific insertions in the helicase module are involved in binding to single-stranded DNA regions, DNA unwinding and supercoiling. Biochemical and structural data suggest that DNA processing by reverse gyrase is not based on sequential action of the helicase and topoisomerase domains, but rather the result of an intricate cooperation of both domains at all stages of the reaction. This review summarizes the recent advances of our understanding of the reverse gyrase mechanism. We put forward and discuss a refined, yet simple model in which reverse gyrase directs strand passage toward increasing linking numbers and positive supercoiling by controlling the conformation of a bound DNA bubble.

| S-EPMC4117796 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data