Diversity of mechanisms in Ras–GAP catalysis of guanosine triphosphate hydrolysis revealed by molecular modeling

2019 ◽  
Vol 17 (19) ◽  
pp. 4879-4891 ◽  
Author(s):  
Bella L. Grigorenko ◽  
Ekaterina D. Kots ◽  
Alexander V. Nemukhin
Keyword(s):  
Molecular Modeling ◽  
Guanosine Triphosphate ◽  
Gtp Hydrolysis

Different mechanisms of GTP hydrolysis by Ras–GAP are revealed in QM/MM simulations depending on molecular groups at position 61 in Ras.

scholarly journals Regulation of Sar1 NH2 terminus by GTP binding and hydrolysis promotes membrane deformation to control COPII vesicle fission

2005 ◽  
Vol 171 (6) ◽  
pp. 919-924 ◽  
Author(s):  
Anna Bielli ◽  
Charles J. Haney ◽  
Gavin Gabreski ◽  
Simon C. Watkins ◽  
Sergei I. Bannykh ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Binding ◽  
Guanosine Triphosphate ◽  
Amphipathic Peptide ◽  
Gtp Hydrolysis ◽  
Membrane Deformation ◽  
Gtp Binding ◽  
Copii Vesicle ◽  
Copii Vesicles ◽  
And Control

The mechanisms by which the coat complex II (COPII) coat mediates membrane deformation and vesicle fission are unknown. Sar1 is a structural component of the membrane-binding inner layer of COPII (Bi, X., R.A. Corpina, and J. Goldberg. 2002. Nature. 419:271–277). Using model liposomes we found that Sar1 uses GTP-regulated exposure of its NH2-terminal tail, an amphipathic peptide domain, to bind, deform, constrict, and destabilize membranes. Although Sar1 activation leads to constriction of endoplasmic reticulum (ER) membranes, progression to effective vesicle fission requires a functional Sar1 NH2 terminus and guanosine triphosphate (GTP) hydrolysis. Inhibition of Sar1 GTP hydrolysis, which stabilizes Sar1 membrane binding, resulted in the formation of coated COPII vesicles that fail to detach from the ER. Thus Sar1-mediated GTP binding and hydrolysis regulates the NH2-terminal tail to perturb membrane packing, promote membrane deformation, and control vesicle fission.

scholarly journals Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase

2008 ◽  
Vol 182 (6) ◽  
pp. 1141-1151 ◽  
Author(s):  
Christopher L. Brett ◽  
Rachael L. Plemel ◽  
Braden T. Lobingier ◽  
Marissa Vignali ◽  
Stanley Fields ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signal Propagation ◽  
Rab Gtpase ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Guanosine Triphosphate ◽  
Casein Kinase I ◽  
Gtp Hydrolysis ◽  
Nucleotide Exchange Factor

Rab guanosine triphosphatases (GTPases) are pivotal regulators of membrane identity and dynamics, but the in vivo pathways that control Rab signaling are poorly defined. Here, we show that the GTPase-activating protein Gyp7 inactivates the yeast vacuole Rab Ypt7 in vivo. To efficiently terminate Ypt7 signaling, Gyp7 requires downstream assistance from an inhibitory casein kinase I, Yck3. Yck3 mediates phosphorylation of at least two Ypt7 signaling targets: a tether, the Vps-C/homotypic fusion and vacuole protein sorting (HOPS) subunit Vps41, and a SNARE, Vam3. Phosphorylation of both substrates is opposed by Ypt7-guanosine triphosphate (GTP). We further demonstrate that Ypt7 binds not one but two Vps-C/HOPS subunits: Vps39, a putative Ypt7 nucleotide exchange factor, and Vps41. Gyp7-stimulated GTP hydrolysis on Ypt7 therefore appears to trigger both passive termination of Ypt7 signaling and active kinase-mediated inhibition of Ypt7's downstream targets. We propose that signal propagation through the Ypt7 pathway is controlled by integrated feedback and feed-forward loops. In this model, Yck3 enforces a requirement for the activated Rab in docking and fusion.

scholarly journals Rab17 regulates apical delivery of hepatic transcytotic vesicles

2018 ◽  
Vol 29 (23) ◽  
pp. 2887-2897 ◽  
Author(s):  
Anneliese C. Striz ◽  
Anna P. Stephan ◽  
Alfonso López-Coral ◽  
Pamela L. Tuma
Keyword(s):  
Dominant Negative ◽  
Apical Surface ◽  
Wild Type ◽  
Major Focus ◽  
Guanosine Triphosphate ◽  
Gtp Hydrolysis ◽  
Vesicle Docking ◽  
Guanosine Diphosphate ◽  
Selective Interactions ◽  
General Component

A major focus for our laboratory is identifying the molecules and mechanisms that regulate basolateral-to-apical transcytosis in polarized hepatocytes. Our most recent studies have focused on characterizing the biochemical and functional properties of the small rab17 GTPase. We determined that rab17 is a monosumoylated protein and that this modification likely mediates selective interactions with the apically located syntaxin 2. Using polarized hepatic WIF-B cells exogenously expressing wild-type, dominant active/guanosine triphosphate (GTP)-bound, dominant negative/guanosine diphosphate (GDP)-bound, or sumoylation-deficient/K68R rab17 proteins, we confirmed that rab17 regulates basolateral-to-apical transcytotic vesicle docking and fusion with the apical surface. We further confirmed that transcytosis is impaired from the subapical compartment to the apical surface and that GTP-bound and sumoylated rab17 are likely required for apical vesicle docking. Because expression of the GTP-bound rab17 led to impaired transcytosis, whereas wild type had no effect, we further propose that rab17 GTP hydrolysis is required for vesicle delivery. We also determined that transcytosis of three classes of newly synthesized apical residents showed similar responses to rab17 mutant expression, indicating that rab17 is a general component of the transcytotic machinery required for apically destined vesicle docking and fusion.

scholarly journals Conformational changes in tubulin in GMPCPP and GDP-taxol microtubules observed by cryoelectron microscopy

2012 ◽  
Vol 198 (3) ◽  
pp. 315-322 ◽  
Author(s):  
Hiroaki Yajima ◽  
Toshihiko Ogura ◽  
Ryo Nitta ◽  
Yasushi Okada ◽  
Chikara Sato ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Regulatory Mechanism ◽  
Reconstruction Algorithm ◽  
Microtubule Dynamics ◽  
Structural Basis ◽  
Cryoelectron Microscopy ◽  
Guanosine Triphosphate ◽  
Gtp Hydrolysis ◽  
Guanosine Diphosphate ◽  
The Stability

Microtubules are dynamic polymers that stochastically switch between growing and shrinking phases. Microtubule dynamics are regulated by guanosine triphosphate (GTP) hydrolysis by β-tubulin, but the mechanism of this regulation remains elusive because high-resolution microtubule structures have only been revealed for the guanosine diphosphate (GDP) state. In this paper, we solved the cryoelectron microscopy (cryo-EM) structure of microtubule stabilized with a GTP analogue, guanylyl 5′-α,β-methylenediphosphonate (GMPCPP), at 8.8-Å resolution by developing a novel cryo-EM image reconstruction algorithm. In contrast to the crystal structures of GTP-bound tubulin relatives such as γ-tubulin and bacterial tubulins, significant changes were detected between GMPCPP and GDP-taxol microtubules at the contacts between tubulins both along the protofilament and between neighboring protofilaments, contributing to the stability of the microtubule. These findings are consistent with the structural plasticity or lattice model and suggest the structural basis not only for the regulatory mechanism of microtubule dynamics but also for the recognition of the nucleotide state of the microtubule by several microtubule-binding proteins, such as EB1 or kinesin.

scholarly journals Mechanism of Guanosine Triphosphate Hydrolysis by the Visual Proteins Arl3-RP2: Free Energy Reaction Profiles Computed with Ab Initio Type QM/MM Potentials

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3998
Author(s):  
Maria G. Khrenova ◽  
Egor S. Bulavko ◽  
Fedor D. Mulashkin ◽  
Alexander V. Nemukhin
Keyword(s):  
Molecular Dynamics ◽  
Reaction Mechanism ◽  
Ab Initio ◽  
Md Simulations ◽  
Human Vision ◽  
Basis Sets ◽  
Hydrolysis Rate ◽  
Guanosine Triphosphate ◽  
Gtp Hydrolysis ◽  
Energy Profiles

We report the results of calculations of the Gibbs energy profiles of the guanosine triphosphate (GTP) hydrolysis by the Arl3-RP2 protein complex using molecular dynamics (MD) simulations with ab initio type QM/MM potentials. The chemical reaction of GTP hydrolysis to guanosine diphosphate (GDP) and inorganic phosphate (Pi) is catalyzed by GTPases, the enzymes, which are responsible for signal transduction in live cells. A small GTPase Arl3, catalyzing the GTP → GDP reaction in complex with the activating protein RP2, constitute an essential part of the human vision cycle. To simulate the reaction mechanism, a model system is constructed by motifs of the crystal structure of the Arl3-RP2 complexed with a substrate analog. After selection of reaction coordinates, energy profiles for elementary steps along the reaction pathway GTP + H2O → GDP + Pi are computed using the umbrella sampling and umbrella integration procedures. QM/MM MD calculations are carried out, interfacing the molecular dynamics program NAMD and the quantum chemistry program TeraChem. Ab initio type QM(DFT)/MM potentials are computed with atom-centered basis sets 6-31G** and two hybrid functionals (PBE0-D3 and ωB97x-D3) of the density functional theory, describing a large QM subsystem. Results of these simulations of the reaction mechanism are compared to those obtained with QM/MM calculations on the potential energy surface using a similar description of the QM part. We find that both approaches, QM/MM and QM/MM MD, support the mechanism of GTP hydrolysis by GTPases, according to which the catalytic glutamine side chain (Gln71, in this system) actively participates in the reaction. Both approaches distinguish two parts of the reaction: the cleavage of the phosphorus-oxygen bond in GTP coupled with the formation of Pi, and the enzyme regeneration. Newly performed QM/MM MD simulations confirmed the profile predicted in the QM/MM minimum energy calculations, called here the pathway-I, and corrected its relief at the first elementary step from the enzyme–substrate complex. The QM/MM MD simulations also revealed another mechanism at the part of enzyme regeneration leading to pathway-II. Pathway-II is more consistent with the experimental kinetic data of the wild-type complex Arl3-RP2, whereas pathway-I explains the role of the mutation Glu138Gly in RP2 slowing down the hydrolysis rate.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

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