How does ras activate raf

It nonetheless shows that the short linker region plays a role in stabilizing RBD and CRD together as one extended structure. To examine the role of hydrophobic linker region residues in stabilizing the RBDCRD structure, we mutated residue L within the linker region to alanine.

The r. The interactions are colored using the following notation: hydrogen bonds—solid blue lines, salt bridge—solid red lines, non-bonded contacts—striped orange lines width of the striped line is proportional to the number of atomic contacts.

Intermolecular hydrogen bonds are indicated by dashed black lines. Cumulatively, these experiments indicate that when CRD is present as an isolated domain, we do not detect a binding interaction with either the farnesyl group or G-domain of KRAS under the conditions used in our experiments. In CRD, residue M interacts with RBD via hydrophobic interaction, while C interacts with T located in the linker region via hydrophobic interaction and hydrogen bonding with main chain atoms.

Fully conserved residues are highlighted in black. The switch and inter-switch regions are indicated above the alignment. Interestingly, these members of the RAS subfamily have significantly divergent interswitch region sequences Fig. While these mutants retain interaction with RAF1 Fig. The importance of V45 is further highlighted by our sequence alignment, which indicates that while V45 is conserved in RAS isoforms, over half of the other RAS subfamily GTPases have long-chain amino acids glutamate or arginine instead of valine Fig.

Table 1 and Supplementary Table 1. The presence of a larger side chain at G12, G13, or Q61 position results in local rearrangement of side chains of some of the neighboring residues present in the switch regions without perturbing interactions at KRAS-RBDCRD interfaces. The structural superposition was carried out using KRAS residues.

The color scheme for different structures is shown in the panel. In addition, NMR titration and paramagnetic relaxation enhancement experiments involving RAF1 CRD binding to nanodisc showed chemical shift perturbations and PRE-induced peak broadening for basic residues K and K, and hydrophobic residues around them 6 , 8.

The electrostatic surface representation of this region of CRD highlights a hydrophobic and basic patch on the surface that is likely to be involved in the interaction with PS-containing membranes Fig. This model also positions previously identified CRD membrane-interacting residues into the lipid bilayer and may therefore provide a snapshot of RAF1 activation by KRAS at the membrane.

However, in these structures, the entire N-terminal half of BRAF is either not observed or, in the case of the autoinhibited complex, only the CRD can be seen centrally anchored between and the kinase domain of BRAF 5. These two CRD residues are located in the hydrophobic loops, where they play an important role in anchoring CRD to the membrane.

Recent cryoEM structures by Park et al. The CRD of BRAF occupies a central position where its membrane-binding surface is buried by interactions with the dimer and the kinase domain. The molecular handcuffing of BRAF by therefore maintains the autoinhibited state by blocking kinase domain dimerization and preventing CRD from binding to the membrane.

Structures were aligned by their CRD domains. This results in CRD coming out of the autoinhibited conformation and interacting with the membrane and KRAS, and in turn exposing the phosphorylated serine present in the CR2 region. It has been suggested that CRD extraction upon RAS binding and its localization to the membrane is the critical event that triggers the release from the molecular handcuffing caused by the dimer 5.

Our work, and the work of others, has revealed that CRD plays a crucial role in stabilizing both the autoinhibited and active states of RAF. Discrete interfaces on CRD are arranged in a way that allows this domain to stabilize the autoinhibited state by burying membrane-interacting residues, or conversely facilitate the active state by presenting membrane anchoring residues. This role is distinct from its role in binding to RAS, as suggested previously by mutagenesis and biochemical analysis.

Here, we extend these early observations to show that multiple mutations in the interswitch region of RAS or in CRD can result in impaired kinase activation, often with minimal effects on binding.

While RAS has been described as a binary switch, our characterization of CRD indicates that it also plays a binary role in the activation—inactivation cycle of RAF kinases. The distinction between binding and activation may explain why some members of the RAS superfamily fail to fully activate RAF kinases, despite being able to bind to RAF through the highly conserved switch-I region. We speculate that the unique interswitch regions of RAS family members may provide specificity for different effector functions, despite the shared switch-I sequences.

It is also reasonable to expect nonspecific consequences when targeting the RAS-RBD interaction given that this this interface is conserved amongst members of the RAS superfamily. Conversely, targeting the interaction between CRD and RAS, or CRD and the plasma membrane, may offer new therapeutic opportunities, considering their lower binding affinities, the specificity afforded by the interswitch region, and the more flexible nature of the protein interfaces. Constructs for protein expression were produced using Gateway recombination-based cloning as described previously 41 using attB-flanked E.

In all cases, final protein expression constructs were generated by Gateway LR recombination into pDest Addgene , an Escherichia coli T7-based expression vector based on pET42 and incorporating hexa-histidine His6 and maltose-binding protein MBP tags at the N-terminus of the protein. Expression constructs were generated by Gateway LR recombination into pDest, a pET21 variant vector with a T7 promoter, and no fusion tags.

Crystallization hits obtained from initial screening were optimized to improve the diffraction quality by systematically varying the pH, individual component concentrations, and the presence of additive and detergents Supplementary Table 1. KRAS constructs with CS mutation was also used to improve the resolution during screening and optimization as this mutation presumably reduces inadvertent cysteine oxidation during crystallization.

Crystallographic datasets were integrated and scaled using XDS The initial solution obtained from molecular replacement was refined using the program Phenix. The model was further improved using iterative cycles of manual model building in COOT 48 and refinement with Phenix. These solvent molecules were manually checked during model building until the final round of refinement was completed.

Refinement statistics for the structures are summarized in Table 1. The amino acid sequence alignments were carried out using Clustal Omega 50 , and the figures were produced using ESPript Crystallographic and structural analysis software support is provided by the SBGrid consortium Neutravidin Pierce was amine coupled to the carboxymethylated dextran surface of a CM5 sensor chip GE Healthcare using standard amine coupling chemistry.

The CM5 chip surface was first activated with 0. Flow cell 1 was used for referencing and buffer injections were included for referencing purposes. SPR sensorgrams were normalized by the capture level of KRAS to allow direct comparison between different experimental runs.

The supernatant was collected, and the elution was repeated twice, followed by one wash with buffer without peptide to remove traces of eluate from the beads. A fraction of each assay sample was analyzed by Western blot as above. RAF1 proteins in these composite systems are then evaluated for steric overlap with lipids in the membrane associated with the simulated KRAS protein. The number of protein residues that clash with lipids any protein atom within 0.

Orientation-specific ensemble averaging over a total of , simulation snapshots provides Boltzmann-weighted averages over the sampled displacement of the KRAS G-domain from the center of mass of the lipid bilayer along its normal, d Gz. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data supporting the findings of this manuscript are available from the corresponding authors upon request. Source data are provided with this paper. Lavoie, H. Cell Biol. Terrell, E. Ras-mediated activation of the Raf family kinases. Cold Spring Harb. Ostrem, J. Direct small-molecule inhibitors of KRAS: from structural insights to mechanism-based design. Drug Discov. Wellbrock, C. The RAF proteins take centre stage. Park, E. Nature , — Fang, Z. Improta-Brears, T. Mutational analysis of Raf-1 cysteine rich domain: requirement for a cluster of basic aminoacids for interaction with phosphatidylserine.

Cell Biochem. Travers, T. Brtva, T. Two distinct Raf domains mediate interaction with Ras. Daub, M. Finally, Ras does not have an allosteric role for the three effectors discussed above. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Tsai CJ, Nussinov R. PLoS Comput Biol. Liu J, Nussinov R. Allostery: an overview of its history, concepts, methods, and applications. Nussinov R. Introduction to protein ensembles and allostery. Chem Rev. The role of dynamic conformational ensembles in biomolecular recognition.

Nat Chem Biol. Biophys J. The structural basis for Ras activation of PI3Kalpha lipid kinase. Phys Chem Chem Phys. Crystal structure of the PI 3-kinase p85 amino-terminal SH2 domain and its phosphopeptide complexes. Nat Struct Biol. Activation of PI3Kalpha by physiological effectors and by oncogenic mutations: structural and dynamic effects. Biophys Rev.

Ras-mediated activation of the raf family kinases. Cold Spring Harb Perspect Med. Lavoie H, Therrien M. Nat Rev Mol Cell Biol. Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues. Mol Cell Biol.

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Molecular cloning and characterization of an activated human c-raf-1 gene. Mutational activation of c-raf-1 and definition of the minimal transforming sequence. This is not the case for the very stable Ras—RBD complex. In the presence of Ras, Raf is most highly populated in the Ras-bound state due to a shift of the free state fraction.

The equilibrium between the autoinhibited state and the free state will then be restored by a certain shift of the autoinhibited state to the free state. Kinase domain dimerization can take place even in the absence of Ras; however, GTP-bound active Ras raises the otherwise low population of the active species, with the exposed kinase domain prepped for dimerization. Thus, even though the mechanisms of Ras activation of its effectors differ, in none of those explored here allostery is incurred by Ras action.

Below we provide the mechanistic details. Alternatively, EGFR overexpression can take place. The RTK motif already accomplishes recruitment to the membrane with the coupled conformational change that relieves the autoinhibition and switches it from the inactive to the active state.

The conformational change created by Ras binding is insignificant, and unlikely to play a role in activation. We conclude that Ras binding serves to further increase the PI3K residence time at the membrane, stabilizing and facilitating PIP 2 binding at the active site. Raf is a multidomain protein. It has a variable length N-terminal tail that was proposed to mediate calcium-dependent B-Raf homo- and hetero-dimerization 60 , interact with the C-terminal 61 , and be responsible for A-Raf low basal activity.

In the inactive state, monomeric Raf is autoinhibited. It's likely autoinhibited organization has recently been reviewed 9 along with the supporting experimental data and theoretical considerations 11 , 61 — This is not the case for the HRas farnesyl group. However, different than KRas, HRas has also two palmitoyls, and the two membrane-anchored palmitoyls lend stability to the system Additional interaction details of the different Ras—Raf systems have also been uncovered 59 , 89 , 94 — In a favored orientation, KRas4B attaches to the membrane through its farnesylated hypervariable region HVR in a way such that the effector binding site faces away from the membrane and is largely exposed.

This permits the RBD to interact at the effector binding site while the CRD is anchored at the membrane through its loop. The nanomolar affinity of the Ras—RBD interaction has been measured in solution. However, under physiological conditions at the membrane, fluctuations that take place and molecular dynamics MD simulations indicate that these can be significant. The enhanced affinity promotes a population shift of the Raf ensemble toward this Ras-bound state, relieving the autoinhibition.

High affinity is not the sole factor controlling the relief of Raf's autoinhibition and population shift toward the open state. Ser phosphorylation of B-Raf weakens the autoinhibition; phosphorylated Ser of Raf-1 is recognized by proteins 86 , 87 , 97 , 98 , promoting the autoinhibition. Dephosphorylation by protein phosphatase 2A PP2A and protein phosphatase 1 PP1 releases it, shifting the equilibrium toward open state 11 , 80 , 99 — The interaction of the N-terminal with the kinase domain is likely to be weak 9.

Simultaneous binding at both sites can promote the autoinhibited state by stabilizing the interaction of the N-terminal segment and the kinase domain 11 , 73 , 87 , — Taken together, this raises the question of why long linkers?

The membrane is crowded. The linker efficiently connects the protein assemblies at the cytoplasm with signals communicated through receptor proteins, such as RTKs. In the cytoplasm, dimers of Raf kinase domains gather in large complexes, including mitogen-activated protein kinase MEK and extracellular signal-regulated kinase ERK dimers.

Large scaffolding and adaptor proteins are also involved, e. The long linker provides an effective and pragmatic solution, enabling formation of clusters in the cytoplasm thus signaling efficiency. The long linkers also vacate the requirement for Ras dimerization for Raf's activation. They allow Ras nanoclusters-mediated Raf's dimerization and activation Figure 1. Thus, rather than allostery, current data argues for a shift of the ensemble through release of the autoinhibited, closed state.

In the absence of active Ras molecules, Raf mostly populates a closed autoinhibited state, with access to the kinase domain hindered by other segments. This mechanism is also supported by the dual interaction, phosphorylation dephosphorylation experiments and mutational data [e. It can explain why Raf evolved tight interaction with Ras and why Ras nanoclusters can function effectively in Raf's activation It can also clarify how the large Raf assemblies with MAPK kinases and scaffolding proteins can form, act efficiently , and allow signaling dynamics despite the crowded membrane surface.

Overexpression of YAP1 induces cell proliferation The linker between the two domains is short 5 residues and contains a flexible hinge. In the presence of active Ras, the equilibrium shifts in favor of the tight Ras—RA interaction. This shift in the MST ensemble from the inactive closed state to the open state permits kinase domain homodimerization and activation via trans-autophosphorylation.

Conformational ensembles and their shifts underlie biological processes 1 — 4 , 30 , 32 , — Population shifts between two states due to differences in the stabilities follow the thermodynamic rule that systems are always driven to their free energy minima.

Understanding how Ras effectors are regulated is of paramount importance since it can help in pharmacological discovery. Scenarios involving high affinity to Ras and long disordered interdomain linkers are likely to discourage allosteric transmission. A tell-tale is the presence or absence of observable conformational changes 27 , If binding promotes a conformational change, allostery is likely at play Figure 2.

Finally, Ras does not have an allosteric role for the three effectors discussed above. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

National Center for Biotechnology Information , U. Journal List Front Oncol v. Front Oncol. Published online Nov Author information Article notes Copyright and License information Disclaimer. This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology.

Received Jul 18; Accepted Oct The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. This article has been cited by other articles in PMC. Abstract The mechanism through which oncogenic Ras activates its effectors is vastly important to resolve.

Introduction Is allostery driving Ras activation of its effectors? Open in a separate window. Figure 1. Allosteric Activation: Definition and Background Classically, allosteric activation is defined as inducing a conformational change in the active site of the enzyme by binding at a location other than the active site.

Figure 2. Concluding Remarks Conformational ensembles and their shifts underlie biological processes 1 — 4 , 30 , 32 , — Author Contributions All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Footnotes Funding. References 1. Tsai CJ, Nussinov R. PLoS Comput Biol. Liu J, Nussinov R. Allostery: an overview of its history, concepts, methods, and applications. Nussinov R. Introduction to protein ensembles and allostery. Chem Rev. The role of dynamic conformational ensembles in biomolecular recognition.

Nat Chem Biol. Biophys J. The structural basis for Ras activation of PI3Kalpha lipid kinase. Phys Chem Chem Phys. Crystal structure of the PI 3-kinase p85 amino-terminal SH2 domain and its phosphopeptide complexes. Nat Struct Biol. Activation of PI3Kalpha by physiological effectors and by oncogenic mutations: structural and dynamic effects. Biophys Rev. Ras-mediated activation of the raf family kinases.

Cold Spring Harb Perspect Med. Lavoie H, Therrien M. Nat Rev Mol Cell Biol. Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues.

Mol Cell Biol. Quantitative analysis of the complex between p21ras and the Ras-binding domain of the human Raf-1 protein kinase. J Biol Chem.

Acta Crystallogr D Biol Crystallogr. The mechanism of PI3Kalpha activation at the atomic level. Chem Sci. A direct interaction between oncogenic Ha-Ras and phosphatidylinositol 3-kinase is not required for Ha-Ras-dependent transformation of epithelial cells. Curr Opin Struct Biol.