Nucleotide Binding Switches the Information Flow in Ras GTPases

The Ras superfamily comprises many guanine nucleotide-binding proteins (G proteins) that are essential to intracellular signal transduction. The guanine nucleotide-dependent intrinsic flexibility patterns of five G proteins were investigated in atomic detail through Molecular Dynamics simulations of the GDP- and GTP-bound states (SGDP and SGTP, respectively). For all the considered systems, the intrinsic flexibility of SGDP was higher than that of SGTP, suggesting that Guanine Exchange Factor (GEF) recognition and nucleotide switch require higher amplitude motions than effector recognition or GTP hydrolysis. Functional mode, dynamic domain, and interaction energy correlation analyses highlighted significant differences in the dynamics of small G proteins and Gα proteins, especially in the inactive state. Indeed, SGDP of Gαt, is characterized by a more extensive energy coupling between nucleotide binding site and distal regions involved in GEF recognition compared to small G proteins, which attenuates in the active state. Moreover, mechanically distinct domains implicated in nucleotide switch could be detected in the presence of GDP but not in the presence of GTP. Finally, in small G proteins, functional modes are more detectable in the inactive state than in the active one and involve changes in solvent exposure of two highly conserved amino acids in switches I and II involved in GEF recognition. The average solvent exposure of these amino acids correlates in turn with the rate of GDP release, suggesting for them either direct or indirect roles in the process of nucleotide switch. Collectively, nucleotide binding changes the information flow through the conserved Ras-like domain, where GDP enhances the flexibility of mechanically distinct portions involved in nucleotide switch, and favors long distance allosteric communication (in Gα proteins), compared to GTP

From Unspecific Quenching to Specific Signaling: Functional GTPase Assays Utilizing Quenching Resonance Energy Transfer (QRET) Technology

The aim of the work presented in this study was to demonstrate the wide applicability of a single-label quenching resonance energy transfer (QRET) assay based on time-resolved lanthanide luminescence. QRET technology is proximity dependent method utilizing weak and unspecific interaction between soluble quencher molecule and lanthanide chelate. The interaction between quencher and chelate is lost when the ligand binds to its target molecule. The properties of QRET technology are especially useful in high throughput screening (HTS) assays. At the beginning of this study, only end-point type QRET technology was available. To enable efficient study of enzymatic reactions, the QRET technology was further developed to enable measurement of reaction kinetics. This was performed using proteindeoxyribonuclei acid (DNA) interaction as a first tool to monitor reaction kinetics. Later, the QRET was used to study nucleotide exchange reaction kinetics and mutation induced effects to the small GTPase activity. Small GTPases act as a molecular switch shifting between active GTP bound and inactive GDP bound conformation. The possibility of monitoring reaction kinetics using the QRET technology was evaluated using two homogeneous assays: a direct growth factor detection assay and a nucleotide exchange monitoring assay with small GTPases. To complete the list, a heterogeneous assay for monitoring GTP hydrolysis using small GTPases, was developed. All these small GTPase assays could be performed using nanomolar protein concentrations without GTPase pretreatment. The results from these studies demonstrated that QRET technology can be used to monitor reaction kinetics and further enable the possibility to use the same method for screening.Siirretty Doriast

Structural communication between the GTPase Sec4p and its activator Sec2p: Determinants of GEF activity and early deformations to nucleotide release

Ras GTPases are molecular switches that cycle between OFF and ON states depending on the bound nucleotide (i.e. GDP-bound and GTP-bound, respectively).The Rab GTPase, Sec4p, plays regulatory roles in multiple steps of intracellular vesicle trafficking. Nucleotide release is catalyzed by the Guanine Nucleotide Exchange Factor (GEF) Sec2p.Here, the integration of structural information with molecular dynamics (MD) simulations addressed a number of questions concerning the intrinsic and stimulated dynamics of Sec2p and Sec4p as well as the chain of structural deformations leading to GEF-assisted activation of the Rab GTPase.Sec2p holds an intrinsic ability to adopt the conformation found in the crystallographic complexes with Sec4p, thus suggesting that the latter selects and shifts the conformational equilibrium towards a pre-existing bound-like conformation of Sec2p.The anchoring of Sec4p to a suitable conformation of Sec2p favors the Sec2p-assisted pulling on itself of the a1/switch 1 (SWI) loop and of SWI, which loose any contact with GDP. Those deformations of Sec4p would occur earlier. Formation of the final Sec2p-Sec4p hydrophobic interface, accomplishes later. Disruption of the nucleotide cage would cause firstly loss of interactions with the guanine ring and sec-ondly loss of interactions with the phosphates.The ease in sampling the energy landscape and adopting a bound-like conformation likely favors the catalyzing ability of GEFs for Ras GTPases.(c) 2022 Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Bio-technology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/)

Structural communication between the GTPase Sec4p and its activator Sec2p: Determinants of GEF activity and early deformations to nucleotide release

Ras GTPases are molecular switches that cycle between OFF and ON states depending on the bound nucleotide (i.e. GDP-bound and GTP-bound, respectively). The Rab GTPase, Sec4p, plays regulatory roles in multiple steps of intracellular vesicle trafficking. Nucleotide release is catalyzed by the Guanine Nucleotide Exchange Factor (GEF) Sec2p. Here, the integration of structural information with molecular dynamics (MD) simulations addressed a number of questions concerning the intrinsic and stimulated dynamics of Sec2p and Sec4p as well as the chain of structural deformations leading to GEF-assisted activation of the Rab GTPase. Sec2p holds an intrinsic ability to adopt the conformation found in the crystallographic complexes with Sec4p, thus suggesting that the latter selects and shifts the conformational equilibrium towards a pre-existing bound-like conformation of Sec2p. The anchoring of Sec4p to a suitable conformation of Sec2p favors the Sec2p-assisted pulling on itself of the α1/switch 1 (SWI) loop and of SWI, which loose any contact with GDP. Those deformations of Sec4p would occur earlier. Formation of the final Sec2p-Sec4p hydrophobic interface, accomplishes later. Disruption of the nucleotide cage would cause firstly loss of interactions with the guanine ring and secondly loss of interactions with the phosphates. The ease in sampling the energy landscape and adopting a bound-like conformation likely favors the catalyzing ability of GEFs for Ras GTPases

The coordination of cell growth during fission yeast mating requires Ras1-GTP hydrolysis

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity

Characterization of a Variant of Tuberous Sclerosis Complex 2 and its Interaction with Rheb

Protein-protein interactions are vital in maintaining proper function and homeostasis in cells. Some signaling pathways are regulated by G-proteins that work like switches to activate and deactivate pathways. Mutations in these proteins, their effectors or the interaction between proteins may cause dysregulation of signals that can lead to many diseases. Rheb, Ras homology enriched in brain, is a Ras family GTPase that is vital in regulation of the mTOR (mammalian target of rapamycin) pathway that signals cell proliferation and growth. Due to the low intrinsic GTPase activity of Rheb, a GTPase activating protein (GAP), Tuberous Sclerosis Complex 2 (TSC2) down regulates Rheb by enhancing its GTPase activity. Currently, very little information is available about TSC2 structures and the molecular details of the interaction between the two proteins. We explored various biochemical and biophysical information of Rheb-TSC2 interaction. In addition, we characterized the stability of the variant, TSC2-218 (D74A), using a 218 amino acid truncated construct of TSC2, and the effects of the single point mutation on the interactions with Rheb. In comparison to the WT, the D74A variant showed to maintain protein stability (thermal and chemical), an increase in secondary (alpha helical) structure, binding and GAP activity towards Rheb

Role of Rho GTPases During Primordial Germ Cell Migration in Zebrafish

Cell migration is an essential biological process during development, adult homeostasis, and disease. Actin cytoskeleton plays crucial role in generating forces required for cell migration. Rho GTPases are key regulators of actin cytoskeleton remodeling during cell migration. To understand the function of Rho GTPases it is important to have the information regarding their spatial-temporal activation pattern in migrating cell. Accessibility of cells migrating in vivo has proven to be a major challenge in studying molecular mechanisms underlying cell migration in their natural milieu. Primordial germ cells (PGCs) in zebrafish arise at distinct positions of the developing embryo and migrate towards their target guided by the chemokine SDF-1a. PGC migration in zebrafish is an excellent in vivo model system to study cell migration processes in vertebrates. It was shown that PGC migrate by forming blebs at their leading edge. The present work focuses at elucidating the role of Rho GTPases in actin cytoskeleton remodeling during PGC migration in zebrafish. To this end I studied actin cytoskeleton dynamics in the presence of mutant forms for three important Rho GTPases: Rac, Cdc42 and RhoA. To study the spatial-temporal activation pattern for Rho GTPases in migrating PGCs, I optimized ratio imaging with FRET biosensors in zebrafish embryo. Utilizing FRET biosensors I showed that Rac and RhoA activities are elevated at the front of the migrating PGC, while Cdc42 is active in the nucleus. Imaging actin cytoskeleton in PGCs revealed that depending on Rac activity, actin accumulates at the front of the cell forming a dense network, which then flows backwards in the process that requires RhoA activity. I propose that in migrating PGCs, Rac function promotes actin accumulation at the front of the cell, while RhoA mediates the actin backward flow thereby generating forces essential for cell locomotion

PAKing up to the endothelium

Angiogenesis recapitulates the growth of blood vessels that progressively expand and remodel into a highly organized and stereotyped vascular network. During adulthood, endothelial cells that formed the vascular wall retain their plasticity and can be engaged in neo-vascularization in response to physiological stimuli, such as hypoxia, wound healing and tissue repair, ovarian cycle and pregnancy. In addition, numerous human diseases and pathological conditions are characterized by an excessive, uncontrolled and aberrant angiogenesis. The signalling pathways involving the small Rho GTPase, Rac and its downstream effector the p21-activated serine/threonine kinase (PAK) had recently emerged as pleiotropic modulators in these processes. Indeed, Rac and PAK were found to modulate endothelial cell biology, such as sprouting, migration, polarity, proliferation, lumen formation, and maturation. Elucidating the Rac/PAK molecular circuitry will provide essential information for the development of new therapeutic agents designed to normalize the blood vasculature in human diseases.Comment: Cell Signal (2009) epub ahead of prin