scholarly journals Membrane surface recognition by the ASAP1 PH domain and consequences for interactions with the small GTPase Arf1

2020 ◽  
Vol 6 (40) ◽  
pp. eabd1882 ◽  
Author(s):  
Olivier Soubias ◽  
Shashank Pant ◽  
Frank Heinrich ◽  
Yue Zhang ◽  
Neeladri Sekhar Roy ◽  
...  
Keyword(s):  
Membrane Surface ◽  
Adenosine Diphosphate ◽  
Small Gtpase ◽  
Dynamics Simulation ◽  
Ph Domain ◽  
Resonance Neutron ◽  
Gtp Hydrolysis ◽  
Surface Recognition ◽  
Guanosine Triphosphatase ◽  
Domain I

Adenosine diphosphate–ribosylation factor (Arf) guanosine triphosphatase–activating proteins (GAPs) are enzymes that need to bind to membranes to catalyze the hydrolysis of guanosine triphosphate (GTP) bound to the small GTP-binding protein Arf. Binding of the pleckstrin homology (PH) domain of the ArfGAP With SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) to membranes containing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is key for maximum GTP hydrolysis but not fully understood. By combining nuclear magnetic resonance, neutron reflectometry, and molecular dynamics simulation, we show that binding of multiple PI(4,5)P2 molecules to the ASAP1 PH domain (i) triggers a functionally relevant allosteric conformational switch and (ii) maintains the PH domain in a well-defined orientation, allowing critical contacts with an Arf1 mimic to occur. Our model provides a framework to understand how binding of the ASAP1 PH domain to PI(4,5)P2 at the membrane may play a role in the regulation of ASAP1.

scholarly journals Membrane Surface Recognition by the ASAP1 Ph Domain and Consequences for Interactions with the Small GTPASE ARF1

2021 ◽  
Vol 120 (3) ◽  
pp. 110a
Author(s):  
Olivier Soubias ◽  
Shashank Pant ◽  
Frank Heinrich ◽  
Yue Zhang ◽  
Paul Randazzo ◽  
...  
Keyword(s):  
Membrane Surface ◽  
Small Gtpase ◽  
Ph Domain ◽  
Surface Recognition

scholarly journals The small GTPases ARL-13 and ARL-3 coordinate intraflagellar transport and ciliogenesis

2010 ◽  
Vol 189 (6) ◽  
pp. 1039-1051 ◽  
Author(s):  
Yujie Li ◽  
Qing Wei ◽  
Yuxia Zhang ◽  
Kun Ling ◽  
Jinghua Hu
Keyword(s):  
Intraflagellar Transport ◽  
Adenosine Diphosphate ◽  
Joubert Syndrome ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Causal Gene ◽  
Guanosine Triphosphatase ◽  
Bidirectional Movement ◽  
Dependent Pathway

Intraflagellar transport (IFT) machinery mediates the bidirectional movement of cargos that are required for the assembly and maintenance of cilia. However, little is known about how IFT is regulated in vivo. In this study, we show that the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor–like protein 13 (ARL-13) encoded by the Caenorhabditis elegans homologue of the human Joubert syndrome causal gene ARL13B, localizes exclusively to the doublet segment of the cilium. arl-13 mutants have shortened cilia with various ultrastructural deformities and a disrupted association between IFT subcomplexes A and B. Intriguingly, depletion of ARL-3, another ciliary small GTPase, partially suppresses ciliogenesis defects in arl-13 mutants by indirectly restoring binding between IFT subcomplexes A and B. Rescue of arl-13 mutants by ARL-3 depletion is mediated by an HDAC6 deacetylase-dependent pathway. Thus, we propose that two conserved small GTPases, ARL-13 and ARL-3, coordinate to regulate IFT and that perturbing this balance results in cilia deformation.

scholarly journals Differential roles of ArfGAP1, ArfGAP2, and ArfGAP3 in COPI trafficking

2008 ◽  
Vol 183 (4) ◽  
pp. 725-735 ◽  
Author(s):  
Carolin Weimer ◽  
Rainer Beck ◽  
Priska Eckert ◽  
Ingeborg Reckmann ◽  
Jörg Moelleken ◽  
...  
Keyword(s):  
Coat Protein ◽  
Protein Complex ◽  
Complex I ◽  
Adenosine Diphosphate ◽  
General Function ◽  
Golgi Membrane ◽  
Gtpase Activating Protein ◽  
Gtp Hydrolysis ◽  
Copi Vesicle ◽  
Guanosine Triphosphatase

The formation of coat protein complex I (COPI)–coated vesicles is regulated by the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor 1 (Arf1), which in its GTP-bound form recruits coatomer to the Golgi membrane. Arf GTPase-activating protein (GAP) catalyzed GTP hydrolysis in Arf1 triggers uncoating and is required for uptake of cargo molecules into vesicles. Three mammalian ArfGAPs are involved in COPI vesicle trafficking; however, their individual functions remain obscure. ArfGAP1 binds to membranes depending on their curvature. In this study, we show that ArfGAP2 and ArfGAP3 do not bind directly to membranes but are recruited via interactions with coatomer. In the presence of coatomer, ArfGAP2 and ArfGAP3 activities are comparable with or even higher than ArfGAP1 activity. Although previously speculated, our results now demonstrate a function for coatomer in ArfGAP-catalyzed GTP hydrolysis by Arf1. We suggest that ArfGAP2 and ArfGAP3 are coat protein–dependent ArfGAPs, whereas ArfGAP1 has a more general function.

scholarly journals RASAL3 preferentially stimulates GTP hydrolysis of the Rho family small GTPase Rac2

2018 ◽  
Author(s):  
Yoonjae Shin ◽  
Yong Kim ◽  
Hyemin Kim ◽  
Nakyoung Shin ◽  
Tae Kim ◽  
...  
Keyword(s):  
Small Gtpase ◽  
Gtp Hydrolysis ◽  
Rho Family ◽  
Hydrolysis Of

scholarly journals ArhGAP15, a Rac-specific GTPase-activating Protein, Plays a Dual Role in Inhibiting Small GTPase Signaling

2013 ◽  
Vol 288 (29) ◽  
pp. 21117-21125 ◽  
Author(s):  
Maria Radu ◽  
Sonali J. Rawat ◽  
Alexander Beeser ◽  
Anton Iliuk ◽  
Weiguo Andy Tao ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Protein Microarray ◽  
Small Gtpases ◽  
Dual Role ◽  
Small Gtpase ◽  
Ph Domain ◽  
Gtpase Activating Protein ◽  
Mutual Inhibition ◽  
Negative Role ◽  
Pak Kinase

Signaling from small GTPases is a tightly regulated process. In this work we used a protein microarray screen to identify the Rac-specific GAP, ArhGAP15, as a substrate of the Rac effectors Pak1 and Pak2. In addition to serving as a substrate of Pak1/2, we found that ArhGAP15, via its PH domain, bound to these kinases. The association of ArhGAP15 to Pak1/2 resulted in mutual inhibition of GAP and kinase catalytic activity, respectively. Knock-down of ArhGAP15 resulted in activation of Pak1/2, both indirectly, as a result of Rac activation, and directly, as a result of disruption of the ArhGAP15/Pak complex. Our data suggest that ArhGAP15 plays a dual negative role in regulating small GTPase signaling, by acting at the level of the GTPase itself, as well interacting with its effector, Pak kinase.

scholarly journals Elongation factor G initiates translocation through a power stroke

2016 ◽  
Vol 113 (27) ◽  
pp. 7515-7520 ◽  
Author(s):  
Chunlai Chen ◽  
Xiaonan Cui ◽  
John F. Beausang ◽  
Haibo Zhang ◽  
Ian Farrell ◽  
...  
Keyword(s):  
Single Molecule ◽  
Messenger Rna ◽  
Elongation Factor ◽  
Power Stroke ◽  
Elongation Factor G ◽  
Gtp Hydrolysis ◽  
Guanosine Triphosphatase ◽  
Prokaryotic Protein ◽  
Domain Iii ◽  
Factor G

During the translocation step of prokaryotic protein synthesis, elongation factor G (EF-G), a guanosine triphosphatase (GTPase), binds to the ribosomal PRE-translocation (PRE) complex and facilitates movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) by one codon. Energy liberated by EF-G’s GTPase activity is necessary for EF-G to catalyze rapid and precise translocation. Whether this energy is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. Free EF-G, not bound to the ribosome, adopts quite different structures in its GTP and GDP forms. Structures of EF-G on the ribosome have been visualized at various intermediate steps along the translocation pathway, using antibiotics and nonhydolyzable GTP analogs to block translocation and to prolong the dwell time of EF-G on the ribosome. However, the structural dynamics of EF-G bound to the ribosome have not yet been described during normal, uninhibited translocation. Here, we report the rotational motions of EF-G domains during normal translocation detected by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. Our study shows that EF-G has a small (∼10°) global rotational motion relative to the ribosome after GTP hydrolysis that exerts a force to unlock the ribosome. This is followed by a larger rotation within domain III of EF-G before its dissociation from the ribosome.

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 Evidence for Two CRIB Domains in Phospholipase D2 (PLD2) That the Enzyme Uses to Specifically Bind to the Small GTPase Rac2

2011 ◽  
Vol 286 (18) ◽  
pp. 16308-16320 ◽  
Author(s):  
Hong-Juan Peng ◽  
Karen M. Henkels ◽  
Madhu Mahankali ◽  
Mary C. Dinauer ◽  
Julian Gomez-Cambronero
Keyword(s):  
Specific Binding ◽  
Strong Association ◽  
Living Cells ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Deletion Mutants ◽  
Ph Domain ◽  
Phospholipase D2

Phospholipase D (PLD) and small GTPases are vital to cell signaling. We report that the Rac2 and the PLD2 isoforms exist in the cell as a lipase-GTPase complex that enables the two proteins to elicit their respective functionalities. A strong association between the two molecules was demonstrated by co-immunoprecipitation and was confirmed in living cells by FRET with CFP-Rac2 and YFP-PLD2 fluorescent chimeras. We have identified the amino acids in PLD2 that define a specific binding site to Rac2. This site is composed of two CRIB (Cdc42-and Rac-interactive binding) motifs that we have named “CRIB-1” and “CRIB-2” in and around the PH domain in PLD2. Deletion mutants PLD2-ΔCRIB-1/2 negate co-immunoprecipitation with Rac2 and diminish the FRET signal in living cells. The PLD2-Rac2 association was further confirmed in vitro using affinity-purified recombinant proteins. Binding was saturable with an apparent Kd of 3 nm and was diminished with PLD2-ΔCRIB mutants. Furthermore, PLD2 bound more efficiently to Rac2-GTP than to Rac2-GDP or to a GDP-constitutive Rac2-N17 mutant. Increasing concentrations of recombinant Rac2 in vitro and in vivo during cell adhesion inhibit PLD2. Conversely, Rac2 activity is increased in the presence of PLD2-WT but not in PLD2-ΔCRIB. We propose that in activated cells PLD2 affects Rac2 in an initial positive feedback, but as Rac2-GTP accumulates in the cell, this constitutes a “termination signal” leading to PLD2 inactivation.

scholarly journals Activation of GTP hydrolysis in mRNA-tRNA translocation by elongation factor G

2015 ◽  
Vol 1 (4) ◽  
pp. e1500169 ◽  
Author(s):  
Wen Li ◽  
Zheng Liu ◽  
Ravi Kiran Koripella ◽  
Robert Langlois ◽  
Suparna Sanyal ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Elongation Factor G ◽  
Direct Role ◽  
Gtp Hydrolysis ◽  
Ribosomal Subunits ◽  
Trna Translocation ◽  
Gtpase Activation ◽  
Guanosine Triphosphatase ◽  
Factor G

During protein synthesis, elongation of the polypeptide chain by each amino acid is followed by a translocation step in which mRNA and transfer RNA (tRNA) are advanced by one codon. This crucial step is catalyzed by elongation factor G (EF-G), a guanosine triphosphatase (GTPase), and accompanied by a rotation between the two ribosomal subunits. A mutant of EF-G, H91A, renders the factor impaired in guanosine triphosphate (GTP) hydrolysis and thereby stabilizes it on the ribosome. We use cryogenic electron microscopy (cryo-EM) at near-atomic resolution to investigate two complexes formed by EF-G H91A in its GTP state with the ribosome, distinguished by the presence or absence of the intersubunit rotation. Comparison of these two structures argues in favor of a direct role of the conserved histidine in the switch II loop of EF-G in GTPase activation, and explains why GTP hydrolysis cannot proceed with EF-G bound to the unrotated form of the ribosome.

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