scholarly journals Molecular assemblies of the catalytic domain of SOS with KRas and oncogenic mutants

2021 ◽  
Vol 118 (12) ◽  
pp. e2022403118
Author(s):  
Zahra Moghadamchargari ◽  
Mehdi Shirzadeh ◽  
Chang Liu ◽  
Samantha Schrecke ◽  
Charles Packianathan ◽  
...  
Keyword(s):  
Ion Mobility ◽  
Catalytic Domain ◽  
Therapeutic Interventions ◽  
Nucleotide Exchange ◽  
Molecular Assemblies ◽  
Oncogenic Ras ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Son Of Sevenless ◽  
Insight Into

Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras–GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility–mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOScat) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRas. We also find KRasG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its activity. A structure of the KRasG13D•SOScat complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRasG13D–GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas–GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRasG13D•SOScat complexes, underscoring the need for more potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.

scholarly journals A Sos proteomimetic as a pan-Ras inhibitor

2021 ◽  
Vol 118 (18) ◽  
pp. e2101027118
Author(s):  
Seong Ho Hong ◽  
Daniel Y. Yoo ◽  
Louis Conway ◽  
Khyle C. Richards-Corke ◽  
Christopher G. Parker ◽  
...  
Keyword(s):  
Rational Design ◽  
Wide Spectrum ◽  
Ras Signaling ◽  
Nucleotide Exchange ◽  
Oncogenic Ras ◽  
Helix Loop Helix ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Son Of Sevenless ◽  
The Subject

Aberrant Ras signaling is linked to a wide spectrum of hyperproliferative diseases, and components of the signaling pathway, including Ras, have been the subject of intense and ongoing drug discovery efforts. The cellular activity of Ras is modulated by its association with the guanine nucleotide exchange factor Son of sevenless (Sos), and the high-resolution crystal structure of the Ras–Sos complex provides a basis for the rational design of orthosteric Ras ligands. We constructed a synthetic Sos protein mimic that engages the wild-type and oncogenic forms of nucleotide-bound Ras and modulates downstream kinase signaling. The Sos mimic was designed to capture the conformation of the Sos helix–loop–helix motif that makes critical contacts with Ras in its switch region. Chemoproteomic studies illustrate that the proteomimetic engages Ras and other cellular GTPases. The synthetic proteomimetic resists proteolytic degradation and enters cells through macropinocytosis. As such, it is selectively toxic to cancer cells with up-regulated macropinocytosis, including those that feature oncogenic Ras mutations.

scholarly journals Post-Golgi Sec Proteins Are Required for Autophagy in Saccharomyces cerevisiae

2010 ◽  
Vol 21 (13) ◽  
pp. 2257-2269 ◽  
Author(s):  
Jiefei Geng ◽  
Usha Nair ◽  
Kyoko Yasumura-Yorimitsu ◽  
Daniel J. Klionsky
Keyword(s):  
Nucleotide Exchange ◽  
Membrane Flow ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Conditional Mutant ◽  
Autophagy Induction ◽  
Atg Genes ◽  
Response To Environmental Change ◽  
Insight Into ◽  
Assembly Site

In eukaryotic cells, autophagy mediates the degradation of cytosolic contents in response to environmental change. Genetic analyses in fungi have identified over 30 autophagy-related (ATG) genes and provide substantial insight into the molecular mechanism of this process. However, one essential issue that has not been resolved is the origin of the lipids that form the autophagosome, the sequestering vesicle that is critical for autophagy. Here, we report that two post-Golgi proteins, Sec2 and Sec4, are required for autophagy. Sec4 is a Rab family GTPase, and Sec2 is its guanine nucleotide exchange factor. In sec2 and sec4 conditional mutant yeast, the anterograde movement of Atg9, a proposed membrane carrier, is impaired during starvation conditions. Similarly, in the sec2 mutant, Atg8 is inefficiently recruited to the phagophore assembly site, which is involved in autophagosome biogenesis, resulting in the generation of fewer autophagosomes. We propose that following autophagy induction the function of Sec2 and Sec4 are diverted to direct membrane flow to autophagosome formation.

scholarly journals Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence

2015 ◽  
Vol 291 (4) ◽  
pp. 1703-1718 ◽  
Author(s):  
Uybach Vo ◽  
Navratna Vajpai ◽  
Liz Flavell ◽  
Romel Bobby ◽  
Alexander L. Breeze ◽  
...  
Keyword(s):  
Intrinsic Fluorescence ◽  
The Novel ◽  
Nucleotide Exchange ◽  
Allosteric Site ◽  
Site Specific ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Catalytic Sites ◽  
Son Of Sevenless

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly 15N-labeled Ras as well as [13C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.

scholarly journals The guanine-nucleotide-exchange factor BopE from Burkholderia pseudomallei adopts a compact version of the Salmonella SopE/SopE2 fold and undergoes a closed-to-open conformational change upon interaction with Cdc42

2008 ◽  
Vol 411 (3) ◽  
pp. 485-493 ◽  
Author(s):  
Abhishek Upadhyay ◽  
Huan-Lin Wu ◽  
Christopher Williams ◽  
Terry Field ◽  
Edouard E. Galyov ◽  
...  
Keyword(s):  
Conformational Change ◽  
Burkholderia Pseudomallei ◽  
Catalytic Domain ◽  
Guanine Nucleotide Exchange Factor ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Closed Conformation ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis, a severe emerging infection. BopE is a GEF (guanine-nucleotide-exchange factor) for the Rho GTPases Cdc42 (cell division cycle 42) and Rac1. We have determined the structure of BopE catalytic domain (amino acids 78–261) by NMR spectroscopy and it shows that BopE78–261 comprises two three-helix bundles (α1α4α5 and α2α3α6). This fold is similar to that adopted by the BopE homologues SopE and SopE2, which are GEFs from Salmonella. Whereas the two three-helix bundles of SopE78–240 and SopE269–240 form the arms of a ‘Λ’ shape, BopE78–261 adopts a more closed conformation with substantial interactions between the two three-helix bundles. We propose that arginine and proline residues are important in the conformational differences between BopE and SopE/E2. Analysis of the molecular interface in the SopE78–240–Cdc42 complex crystal structure indicates that, in a BopE–Cdc42 interaction, the closed conformation of BopE78–261 would engender steric clashes with the Cdc42 switch regions. This implies that BopE78–261 must undergo a closed-to-open conformational change in order to catalyse guanine nucleotide exchange. In an NMR titration to investigate the BopE78–261–Cdc42 interaction, the appearance of additional peaks per NH for residues in hinge regions of BopE78–261 indicates that BopE78–261 does undergo a closed-to-open conformational change in the presence of Cdc42. The conformational change hypothesis is further supported by substantial improvement of BopE78–261 catalytic efficiency through mutations that favour an open conformation. Requirement for closed-to-open conformational change explains the 10–40-fold lower kcat of BopE compared with SopE and SopE2.

scholarly journals The substrate specificity of the human TRAPPII complex’s Rab-guanine nucleotide exchange factor activity

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Meredith L. Jenkins ◽  
Noah J. Harris ◽  
Udit Dalwadi ◽  
Kaelin D. Fleming ◽  
Daniel S. Ziemianowicz ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Rab Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Binding Interface ◽  
Nucleotide Exchange Factor ◽  
Biochemical Characterisation ◽  
Hydrogen Deuterium ◽  
Insight Into

AbstractThe TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.

scholarly journals Molecular Determinants of the Interaction between Coxsackievirus Protein 3A and Guanine Nucleotide Exchange Factor GBF1

2007 ◽  
Vol 81 (10) ◽  
pp. 5238-5245 ◽  
Author(s):  
Els Wessels ◽  
Daniël Duijsings ◽  
Kjerstin H. W. Lanke ◽  
Willem J. G. Melchers ◽  
Catherine L. Jackson ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Binding Domain ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Molecular Determinants ◽  
N Terminus ◽  
Insight Into

ABSTRACT The 3A protein of coxsackievirus B3 (CVB3), a small membrane protein that forms homodimers, inhibits endoplasmic reticulum-to-Golgi complex transport. Recently, we described the underlying mechanism by showing that the CVB3 3A protein binds to and inhibits the function of GBF1, a guanine nucleotide exchange factor for ADP-ribosylation factor 1 (Arf1), thereby interfering with Arf1-mediated COP-I recruitment. This study was undertaken to gain more insight into the molecular determinants underlying the interaction between 3A and GBF1. Here we show that 3A mutants that have lost the ability to dimerize are no longer able to bind to GBF1 and trap it on membranes. Moreover, we identify a conserved region in the N terminus of 3A that is crucial for GBF1 binding but not for 3A dimerization. Analysis of the binding domain in GBF1 showed that the extreme N terminus, the dimerization/cyclophilin binding domain, and the homology upstream of Sec7 domain are required for the interaction with 3A. In contrast to that of full-length GBF1, overexpression of a GBF1 mutant lacking its extreme N terminus failed to rescue the effects of 3A. Together, these data provide insight into the molecular requirements of the interaction between 3A and GBF1.

scholarly journals CalDAG-GEFI Deficiency in a Family with Symptomatic Heterozygous and Homozygous Carriers of a Likely Pathogenic Variant in RASGRP2

2021 ◽  
Vol 22 (22) ◽  
pp. 12423
Author(s):  
Sara Morais ◽  
Mónica Pereira ◽  
Catarina Lau ◽  
Ana Gonçalves ◽  
Catarina Monteiro ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Phenotypic Expression ◽  
Clinical Manifestations ◽  
Pathogenic Variant ◽  
Nucleotide Exchange ◽  
Pathogenic Variants ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Heterozygous Carriers

RASGRP2 encodes the calcium and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) identified as a Rap1-activating molecule. Pathogenic variants previously identified in RASGRP2 allowed the characterization of CalDAG-GEFI deficiency as a non-syndromic, autosomal recessive platelet function disease. We report on the clinical manifestations and laboratory features of a Portuguese family with a likely pathogenic variant in RASGRP2 (c.999G>C leading to a p.Lys333Asn change in the CDC25 catalytic domain of CalDAG-GEFI) and discuss the contribution of this variant to the disease manifestations. Based on the study of this family with one homozygous patient and five heterozygous carriers and on a critical analysis of the literature, we challenge previous knowledge that CalDAG-GEFI deficiency only manifests in homozygous patients. Our data suggest that at least for the RASGRP2 variant reported herein, there is a phenotypic expression, albeit milder, in heterozygous carriers.

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