scholarly journals Conformational resolution of nucleotide cycling and effector interactions for multiple small GTPases in parallel

2019 ◽  
Author(s):  
Ryan C. Killoran ◽  
Matthew J. Smith
Keyword(s):  
Binding Pocket ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Cellular Processes ◽  
Selective Labelling ◽  
Activation Potential ◽  
Single Complex ◽  
Gtpase Activation ◽  
Ras Isoforms

AbstractSmall GTPase proteins alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes. Numerous GTPases are implicated in oncogenesis, particularly three RAS isoforms HRAS, KRAS and NRAS, and the RHO family GTPase RAC1. Signaling networks comprising small GTPases are highly connected, and there is evidence of direct biochemical crosstalk between the functional G-domains of these proteins. The activation potential of a given GTPase is contingent on a co-dependent interaction with nucleotide and a Mg2+ ion, which bind to individual variants via distinct affinities coordinated by residues in the nucleotide binding pocket. Here, we utilize a selective-labelling strategy coupled with real-time nuclear magnetic resonance (NMR) spectroscopy to monitor nucleotide exchange, GTP hydrolysis and effector interactions of multiple small GTPases in a single complex system. We provide new insight on nucleotide preference and the role of Mg2+ in activating both wild-type and oncogenic mutant enzymes. Multiplexing reveals GEF, GAP and effector binding specificity in mixtures of GTPases and establishes the complete biochemical equivalence of the three related RAS isoforms. This work establishes that direct quantitation of the nucleotide-bound conformation is required to accurately resolve GTPase activation potential, as GTPases such as RALA or the G12C mutant of KRAS demonstrate fast exchange kinetics but have a high affinity for GDP. Further, we propose that the G-domains of small GTPases behave autonomously in solution and nucleotide cycling proceeds independent of protein concentration but is highly impacted by Mg2+ abundance.

scholarly journals Conformational resolution of nucleotide cycling and effector interactions for multiple small GTPases determined in parallel

2019 ◽  
Vol 294 (25) ◽  
pp. 9937-9948 ◽  
Author(s):  
Ryan C. Killoran ◽  
Matthew J. Smith
Keyword(s):  
Binding Pocket ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Activation Potential ◽  
Single Complex ◽  
Ras Isoforms

Small GTPases alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes. Numerous GTPases are implicated in oncogenesis, particularly the three RAS isoforms HRAS, KRAS, and NRAS and the RHO family GTPase RAC1. Signaling networks comprising small GTPases are highly connected, and there is some evidence of direct biochemical cross-talk between their functional G-domains. The activation potential of a given GTPase is contingent on a codependent interaction with the nucleotide and a Mg2+ ion, which bind to individual variants with distinct affinities coordinated by residues in the GTPase nucleotide-binding pocket. Here, we utilized a selective-labeling strategy coupled with real-time NMR spectroscopy to monitor nucleotide exchange, GTP hydrolysis, and effector interactions of multiple small GTPases in a single complex system. We provide insight into nucleotide preference and the role of Mg2+ in activating both WT and oncogenic mutant enzymes. Multiplexing revealed guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP), and effector-binding specificities in mixtures of GTPases and resolved that the three related RAS isoforms are biochemically equivalent. This work establishes that direct quantitation of the nucleotide-bound conformation is required to accurately determine an activation potential for any given GTPase, as small GTPases such as RAS-like proto-oncogene A (RALA) or the G12C mutant of KRAS display fast exchange kinetics but have a high affinity for GDP. Furthermore, we propose that the G-domains of small GTPases behave autonomously in solution and that nucleotide cycling proceeds independently of protein concentration but is highly impacted by Mg2+ abundance.

scholarly journals Analysis of RAS protein interactions in living cells reveals a mechanism for pan-RAS depletion by membrane-targeted RAS binders

2020 ◽  
Vol 117 (22) ◽  
pp. 12121-12130
Author(s):  
Yao-Cheng Li ◽  
Nikki K. Lytle ◽  
Seth T. Gammon ◽  
Luke Wang ◽  
Tikvah K. Hayes ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Developmental Disorders ◽  
Degradation Pathway ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Ras Proteins ◽  
Ras Protein ◽  
Protein Levels ◽  
Ras Family ◽  
Ras Isoforms

HRAS, NRAS, and KRAS4A/KRAS4B comprise the RAS family of small GTPases that regulate signaling pathways controlling cell proliferation, differentiation, and survival. RAS pathway abnormalities cause developmental disorders and cancers. We found that KRAS4B colocalizes on the cell membrane with other RAS isoforms and a subset of prenylated small GTPase family members using a live-cell quantitative split luciferase complementation assay. RAS protein coclustering is mainly mediated by membrane association-facilitated interactions (MAFIs). Using the RAS–RBD (CRAF RAS binding domain) interaction as a model system, we showed that MAFI alone is not sufficient to induce RBD-mediated RAS inhibition. Surprisingly, we discovered that high-affinity membrane-targeted RAS binding proteins inhibit RAS activity and deplete RAS proteins through an autophagosome–lysosome-mediated degradation pathway. Our results provide a mechanism for regulating RAS activity and protein levels, a more detailed understanding of which should lead to therapeutic strategies for inhibiting and depleting oncogenic RAS proteins.

Common mechanisms of catalysis in small and heterotrimeric GTPases and their respective GAPs

2017 ◽  
Vol 398 (5-6) ◽  
pp. 523-533 ◽  
Author(s):  
Klaus Gerwert ◽  
Daniel Mann ◽  
Carsten Kötting
Keyword(s):  
G Protein ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Nucleophilic Attack ◽  
Protein Signaling ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
Molecular Reaction ◽  
External Signals

Abstract GTPases are central switches in cells. Their dysfunctions are involved in severe diseases. The small GTPase Ras regulates cell growth, differentiation and apoptosis by transmitting external signals to the nucleus. In one group of oncogenic mutations, the ‘switch-off’ reaction is inhibited, leading to persistent activation of the signaling pathway. The switch reaction is regulated by GTPase-activating proteins (GAPs), which catalyze GTP hydrolysis in Ras, and by guanine nucleotide exchange factors, which catalyze the exchange of GDP for GTP. Heterotrimeric G-proteins are activated by G-protein coupled receptors and are inactivated by GTP hydrolysis in the Gα subunit. Their GAPs are called regulators of G-protein signaling. In the same way that Ras serves as a prototype for small GTPases, Gαi1 is the most well-studied Gα subunit. By utilizing X-ray structural models, time-resolved infrared-difference spectroscopy, and biomolecular simulations, we elucidated the detailed molecular reaction mechanism of the GTP hydrolysis in Ras and Gαi1. In both proteins, the charge distribution of GTP is driven towards the transition state, and an arginine is precisely positioned to facilitate nucleophilic attack of water. In addition to these mechanistic details of GTP hydrolysis, Ras dimerization as an emerging factor in signal transduction is discussed in this review.

scholarly journals The RhoGEF Trio: A Protein with a Wide Range of Functions in the Vascular Endothelium

2021 ◽  
Vol 22 (18) ◽  
pp. 10168
Author(s):  
Lanette Kempers ◽  
Amber J. M. Driessen ◽  
Jos van Rijssel ◽  
Martijn A. Nolte ◽  
Jaap D. van Buul
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Function ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Endothelial Cell Function ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Wide Range ◽  
Range Of Functions

Many cellular processes are controlled by small GTPases, which can be activated by guanine nucleotide exchange factors (GEFs). The RhoGEF Trio contains two GEF domains that differentially activate the small GTPases such as Rac1/RhoG and RhoA. These small RhoGTPases are mainly involved in the remodeling of the actin cytoskeleton. In the endothelium, they regulate junctional stabilization and play a crucial role in angiogenesis and endothelial barrier integrity. Multiple extracellular signals originating from different vascular processes can influence the activity of Trio and thereby the regulation of the forementioned small GTPases and actin cytoskeleton. This review elucidates how various signals regulate Trio in a distinct manner, resulting in different functional outcomes that are crucial for endothelial cell function in response to inflammation.

scholarly journals Autoactivation of small GTPases by the GEF–effector positive feedback modules

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 1676 ◽  
Author(s):  
Andrew B. Goryachev ◽  
Marcin Leda
Keyword(s):  
Cell Biology ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins ◽  
Recent Developments ◽  
Functional Consequences ◽  
Exciting Area

Small GTPases are organizers of a plethora of cellular processes. The time and place of their activation are tightly controlled by the localization and activation of their regulators, guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Remarkably, in some systems, the upstream regulators of GTPases are also found downstream of their activity. Resulting feedback loops can generate complex spatiotemporal dynamics of GTPases with important functional consequences. Here we discuss the concept of positive autoregulation of small GTPases by the GEF–effector feedback modules and survey recent developments in this exciting area of cell biology.

scholarly journals Cross-Kingdom Activation of Vibrio Toxins by ADP-Ribosylation Factor Family GTPases

2020 ◽  
Vol 202 (24) ◽  
Author(s):  
Alfa Herrera ◽  
Karla J. F. Satchell
Keyword(s):  
Vibrio Vulnificus ◽  
Bacterial Species ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Cell Organelle ◽  
Content Type ◽  
Adp Ribosylation ◽  
Cellular Processes ◽  
Adp Ribosyltransferase ◽  
Adp Ribosylation Factor

ABSTRACT Pathogenic Vibrio species use many different approaches to subvert, attack, and undermine the host response. The toxins they produce are often responsible for the devastating effects associated with their diseases. These toxins target a variety of host proteins, which leads to deleterious effects, including dissolution of cell organelle integrity and inhibition of protein secretion. Becoming increasingly prevalent as cofactors for Vibrio toxins are proteins of the small GTPase families. ADP-ribosylation factor small GTPases (ARFs) in particular are emerging as a common host cofactor necessary for full activation of Vibrio toxins. While ARFs are not the direct target of Vibrio cholerae cholera toxin (CT), ARF binding is required for its optimal activity as an ADP-ribosyltransferase. The makes caterpillars floppy (MCF)-like and the domain X (DmX) effectors of the Vibrio vulnificus multifunctional autoprocessing repeats-in-toxin (MARTX) toxin also both require ARFs to initiate autoprocessing and activation as independent effectors. ARFs are ubiquitously expressed in eukaryotes and are key regulators of many cellular processes, and as such they are ideal cofactors for Vibrio pathogens that infect many host species. In this review, we cover in detail the known Vibrio toxins that use ARFs as cross-kingdom activators to both stimulate and optimize their activity. We further discuss how these contrast to toxins and effectors from other bacterial species that coactivate, stimulate, or directly modify host ARFs as their mechanisms of action.

Osmotic stress activates Rac and Cdc42 in neutrophils: role in hypertonicity-induced actin polymerization

2002 ◽  
Vol 282 (2) ◽  
pp. C271-C279 ◽  
Author(s):  
Alison Lewis ◽  
Caterina Di Ciano ◽  
Ori D. Rotstein ◽  
András Kapus
Keyword(s):  
Actin Polymerization ◽  
Cell Movement ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Phosphatidylinositol 3 Kinase ◽  
Cytoskeleton Remodeling ◽  
Clostridium Difficile Toxin ◽  
Clostridium Difficile Toxin B ◽  
Gtpase Activation ◽  
Fold Activation

Hypertonicity inhibits a variety of neutrophil functions through poorly defined mechanisms. Our earlier studies suggest that osmotically induced actin polymerization and cytoskeleton remodeling is a key component in the hypertonic block of exocytosis and cell movement. To gain insight into the signaling mechanisms underlying the hyperosmotic F-actin response, we investigated whether hypertonicity stimulates Rac and Cdc42 and, if so, whether their activation contributes to the hypertonic rise in F-actin. Using a recently developed pull-down assay that specifically captures the active forms of these small GTPases, we found that hypertonicity caused an ∼2.5- and ∼7.2-fold activation of Rac and Cdc42, respectively. This response was rapid and sustained. Small GTPase activation was not mediated by the osmotic stimulation of Src kinases, heterotrimeric G proteins, or phosphatidylinositol 3-kinase. Interestingly, an increase in intracellular ionic strength was sufficient to activate Rac even in the absence of cell shrinkage. Inhibition of Rac and Cdc42 by Clostridium difficile toxin B substantially reduced but did not abolish the hypertonicity-induced F-actin response. Thus hypertonicity is a potent activator of Rac and Cdc42, and this effect seems to play an important but not exclusive role in the hyperosmolarity-triggered cytoskeleton remodeling.

scholarly journals A non-linear system patterns Rab5 GTPase on the membrane

2019 ◽  
Author(s):  
A Cezanne ◽  
J Lauer ◽  
A Solomatina ◽  
IF Sbalzarini ◽  
M Zerial
Keyword(s):  
Lipid Bilayers ◽  
Lipid Membrane ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Membrane Composition ◽  
Dynamic Interactions ◽  
Universal System ◽  
Non Linear ◽  
Non Linear System

AbstractProteins can self-organize into spatial patterns via non-linear dynamic interactions on cellular membranes. Modelling and simulations have shown that small GTPases can generate patterns by coupling guanine nucleotide exchange factors (GEF) to effector binding, generating a positive feedback of GTPase activation and membrane recruitment. Here, we reconstituted the patterning of the small GTPase Rab5 and its GEF/effector complex Rabex5/Rabaptin5 on supported lipid bilayers as a model system for membrane patterning. We show that there is a “handover” of Rab5 from Rabex5 to Rabaptin5 upon nucleotide exchange. A minimal system consisting of Rab5, RabGDI and a complex of full length Rabex5/Rabaptin5 was necessary to pattern Rab5 into membrane domains. Surprisingly, a lipid membrane composition mimicking that of the early endosome was required for Rab5 patterning. The prevalence of GEF/effector coupling in nature suggests a possible universal system for small GTPase patterning involving both protein and lipid interactions.

scholarly journals Divergent Mechanisms Activating RAS and Small GTPases Through Post-translational Modification

2021 ◽  
Vol 8 ◽  
Author(s):  
Natsuki Osaka ◽  
Yoshihisa Hirota ◽  
Doshun Ito ◽  
Yoshiki Ikeda ◽  
Ryo Kamata ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Guanine Nucleotide Exchange ◽  
Ras Superfamily ◽  
Switch Regions

RAS is a founding member of the RAS superfamily of GTPases. These small 21 kDa proteins function as molecular switches to initialize signaling cascades involved in various cellular processes, including gene expression, cell growth, and differentiation. RAS is activated by GTP loading and deactivated upon GTP hydrolysis to GDP. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) accelerate GTP loading and hydrolysis, respectively. These accessory proteins play a fundamental role in regulating activities of RAS superfamily small GTPase via a conserved guanine binding (G)-domain, which consists of five G motifs. The Switch regions lie within or proximal to the G2 and G3 motifs, and undergo dynamic conformational changes between the GDP-bound “OFF” state and GTP-bound “ON” state. They play an important role in the recognition of regulatory factors (GEFs and GAPs) and effectors. The G4 and G5 motifs are the focus of the present work and lie outside Switch regions. These motifs are responsible for the recognition of the guanine moiety in GTP and GDP, and contain residues that undergo post-translational modifications that underlie new mechanisms of RAS regulation. Post-translational modification within the G4 and G5 motifs activates RAS by populating the GTP-bound “ON” state, either through enhancement of intrinsic guanine nucleotide exchange or impairing GAP-mediated down-regulation. Here, we provide a comprehensive review of post-translational modifications in the RAS G4 and G5 motifs, and describe the role of these modifications in RAS activation as well as potential applications for cancer therapy.

scholarly journals Interactions between Cdc42 and the scaffold protein Scd2: requirement of SH3 domains for GTPase binding

2005 ◽  
Vol 388 (1) ◽  
pp. 177-184 ◽  
Author(s):  
Edward WHEATLEY ◽  
Katrin RITTINGER
Keyword(s):  
Binding Sites ◽  
Sh3 Domain ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Titration Calorimetry ◽  
Nucleotide Exchange ◽  
Sh3 Domains ◽  
Binding Surface ◽  
P21 Activated Kinase ◽  
Conventional Manner

The multi-domain protein Scd2 acts as a scaffold upon which the small GTPase Cdc42 (cell division cycle 42), its nucleotide-exchange factor Scd1 and the p21-activated kinase Shk1 assemble to regulate cell polarity and the mating response in fission yeast. In the present study, we show using isothermal titration calorimetry that Scd2 binds two molecules of active GTP-bound Cdc42 simultaneously, but independently of one another. The two binding sites have significantly different affinities, 21 nM and 3 μM, suggesting that they play distinct roles in the Shk1 signalling network. Each of the Cdc42-binding sites includes one of the SH3 (Src homology 3) domains of Scd2. Our data indicate that complex formation does not occur in a conventional manner via the conserved SH3 domain ligand-binding surface. Neither of the isolated SH3 domains is sufficient to interact with the GTPase, and they both require adjacent regions to either stabilize their conformations or contribute to the formation of the Cdc42-binding surface. Furthermore, we show that there is no evidence for an intramolecular PX–SH3 domain interaction, which could interfere with SH3 domain function. This work suggests that SH3 domains might contribute directly to signalling through small GTPases and thereby adds another aspect to the diverse nature of SH3 domains as protein–protein-interaction modules.

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