GDP Release Preferentially Occurs on the Phosphate Side in Heterotrimeric G-proteins

Louet, Maxime; Martinez, Jean; Floquet, Nicolas

doi:10.1371/journal.pcbi.1002595

Cited by 20 publications

(25 citation statements)

References 31 publications

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“…On the other hand, both D150 and GDP were negative charged, and the binding energy between them was merely −0.02 kcal/mol, indicating that D150 is a residue that can contribute only to the specific binding between G proteins and GDP/GTP. All of these results are in accordance with the recent report of Louet et al 62 …”

Section: Resultssupporting

confidence: 94%

Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery

Feng

et al. 2016

J. Chem. Inf. Model.

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Cannabinoid receptor 2 (CB2), a G protein-coupled receptor (GPCR), is a promising target for the treatment of neuropathic pain, osteoporosis, immune system, cancer, and drug abuse. The lack of an experimental three-dimensional CB2 structure has hindered not only the development of studies of conformational differences between the inactive and active CB2 but also the rational discovery of novel functional compounds targeting CB2. In this work, we constructed models of both inactive and active CB2 by homology modeling. Then we conducted two comparative 100 ns molecular dynamics (MD) simulations on the two systems—the active CB2 bound with both the agonist and G protein and the inactive CB2 bound with inverse agonist—to analyze the conformational difference of CB2 proteins and the key residues involved in molecular recognition. Our results showed that the inactive CB2 and the inverse agonist remained stable during the MD simulation. However, during the MD simulations, we observed dynamical details about the breakdown of the “ionic lock” between R1313.50 and D2406.30 as well as the outward/inward movements of transmembrane domains of the active CB2 that bind with G proteins and agonist (TM5, TM6, and TM7). All of these results are congruent with the experimental data and recent reports. Moreover, our results indicate that W2586.48 in TM6 and residues in TM4 (V1644.56–L1694.61) contribute greatly to the binding of the agonist on the basis of the binding energy decomposition, while residues S180–F183 in extracellular loop 2 (ECL2) may be of importance in recognition of the inverse agonist. Furthermore, pharmacophore modeling and virtual screening were carried out for the inactive and active CB2 models in parallel. Among all 10 hits, two compounds exhibited novel scaffolds and can be used as novel chemical probes for future studies of CB2. Importantly, our studies show that the hits obtained from the inactive CB2 model mainly act as inverse agonist(s) or neutral antagonist(s) at low concentration. Moreover, the hit from the active CB2 model also behaves as a neutral antagonist at low concentration. Our studies provide new insight leading to a better understanding of the structural and conformational differences between two states of CB2 and illuminate the effects of structure on virtual screening and drug design.

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Section: Resultssupporting

confidence: 94%

Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery

Feng

et al. 2016

J. Chem. Inf. Model.

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“…Molecular mechanisms that take place in G␣ i1 have been investigated by numerous studies including structural (5, 34, 60), computational (61), and biochemical (13, 14, 33) assays. In particular, multiple turnover GTPase assays like malachite green or radiometric phosphate tests using [␥-…”

Section: Discussionmentioning

confidence: 99%

Integration of Fourier Transform Infrared Spectroscopy, Fluorescence Spectroscopy, Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism

Schröter¹,

Mann²,

Kötting

et al. 2015

Journal of Biological Chemistry

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Background: Multiple turnover GTPase assays of G␣ are dominated by nucleotide exchange. Results: FTIR elucidates single turnover rates and individual phosphate vibrations. Conclusion: G␣ i1 -R178S is slowed down in single turnover hydrolysis by 2 orders of magnitude, G␣ i1 -Asp 229 and -Asp 231 are key players in Ras-like/all-␣ domain coordination. Significance: With FTIR on G␣ established, detailed information on the reaction mechanism can be obtained.

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“…For example, while the biased potential applied in umbrella sampling could force r(thick_gate) to a value around 8.0, from either outward-facing (r(thick_gate) ∼5.5 Å)) or inward-facing (r(thick_gate) ∼10 Å) structure, extremely long simulation time may be required to relax the rest of the protein. To avoid excessive long relaxation time, targeted molecular dynamics has been applied to obtain intermediate structures for use as starting structures for umbrella sampling [58]. In this study, we use the string method with swarm of trajectories to further relax the path from TMD simulations.…”

Section: Methodsmentioning

confidence: 99%

The Molecular Mechanism of Ion-Dependent Gating in Secondary Transporters

Zhao

Noskov

2013

PLoS Comput Biol

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LeuT-like fold Na-dependent secondary active transporters form a large family of integral membrane proteins that transport various substrates against their concentration gradient across lipid membranes, using the free energy stored in the downhill concentration gradient of sodium ions. These transporters play an active role in synaptic transmission, the delivery of key nutrients, and the maintenance of osmotic pressure inside the cell. It is generally believed that binding of an ion and/or a substrate drives the conformational dynamics of the transporter. However, the exact mechanism for converting ion binding into useful work has yet to be established. Using a multi-dimensional path sampling (string-method) followed by all-atom free energy simulations, we established the principal thermodynamic and kinetic components governing the ion-dependent conformational dynamics of a LeuT-like fold transporter, the sodium/benzyl-hydantoin symporter Mhp1, for an entire conformational cycle. We found that inward-facing and outward-facing states of Mhp1 display nearly the same free energies with an ion absent from the Na2 site conserved across the LeuT-like fold transporters. The barrier separating an apo-state from inward-facing or outward-facing states of the transporter is very low, suggesting stochastic gating in the absence of ion/substrate bound. In contrast, the binding of a Na2 ion shifts the free energy stabilizing the outward-facing state and promoting substrate binding. Our results indicate that ion binding to the Na2 site may also play a key role in the intracellular thin gate dynamics modulation by altering its interactions with the transmembrane helix 5 (TM5). The Potential of Mean Force (PMF) computations for a substrate entrance displays two energy minima that correspond to the locations of the main binding site S1 and proposed allosteric S2 binding site. However, it was found that substrate's binds to the site S1 ∼5 kcal/mol more favorable than that to the site S2 for all studied bound combinations of ions and a substrate.

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GDP Release Preferentially Occurs on the Phosphate Side in Heterotrimeric G-proteins

Cited by 20 publications

References 31 publications

Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery

Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery

Integration of Fourier Transform Infrared Spectroscopy, Fluorescence Spectroscopy, Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism

The Molecular Mechanism of Ion-Dependent Gating in Secondary Transporters

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