A Comparative CEST NMR Study of Slow Conformational Dynamics of Small GTPases Complexed with GTP and GTP Analogues

Long, Dong; Marshall, Christopher B.; Bouvignies, Guillaume; Mazhab-Jafari, Mohammad T.; Smith, Matthew J.; Ikura, Mitsuhiko; Kay, Lewis E.

doi:10.1002/anie.201305434

Cited by 39 publications

(43 citation statements)

References 32 publications

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“…The application of multidimensional NMR spectroscopy to active Ras is challenged by the fast hydrolysis of GTP, and as a result experiments are routinely recorded using stable GTP analogues, for example, GppNHp, GppCH 2 p, and GTPγS. These chemical modifications have, unfortunately, been shown to tilt the protein free energy landscape to varying extents, and thereby alter the intrinsic dynamics of Ras . In an effort to directly probe the dynamics of active Ras using native GTP, chemical exchange saturation transfer (CEST) experiments were previously recorded at 5 °C, which slows down both hydrolysis and conformational exchange .…”

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confidence: 99%

Extending the Lifetime of Native GTP‐Bound Ras for Site‐Resolved NMR Measurements: Quantifying the Allosteric Dynamics

et al. 2019

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Characterization of native GTP-bound Ras is important for an appreciation of its cellular signaling and for the design of inhibitors,which however has been depressed by its intrinsic instability.H erein, an effective approach for extending the lifetime of Ras·GTP samples by exploiting the active role of Son of Sevenless (Sos) is demonstrated that sustains the activated state of Ras.T his approach,c ombined with ap ostprocessing method that suppresses residual Ras·GDP signals,i sa pplied to the site-resolved NMR measurement of the allosteric dynamics of Ras·GTP.The observed network of concerted motions well covers the recently identified allosteric inhibitor-binding pockets,b ut the motions are more confined than those of Ras·GppNHp,advocating the use of native GTP for development of allosteric inhibitors.T he Sos-based approach is anticipated to generally facilitate experiments on active Ras when native GTP is preferred.

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Section: Figurementioning

confidence: 99%

Extending the Lifetime of Native GTP‐Bound Ras for Site‐Resolved NMR Measurements: Quantifying the Allosteric Dynamics

et al. 2019

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“…Residues exhibiting NMR signals on this timescale are not necessarily mobile; rearrangements of neighboring residues can change the chemical environment of the residue being probed too. For even slower timescales, chemical exchange saturation transfer (CEST) provides similar information 33 . In a classical application of CMPG experiments, fitting against a dispersion curve 34 revealed residue-specific populations, exchange rates, and chemical shift differences for the enzyme Dihydrofolate Reductase (DHFR) bound to different ligands mimicking states of its catalytic cycle (Fig.…”

Section: Solution Nmr Signals Are Generated By Multiple Conformationsmentioning

confidence: 99%

Integrative, dynamic structural biology at atomic resolution—it's about time

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Fraser²

2015

Nat Methods

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Biomolecules adopt a dynamic ensemble of conformations, each with the potential to interact with binding partners or perform the chemical reactions required for a multitude of cellular functions. Recent advances in X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, and other techniques are helping us realize the dream of seeing—in atomic detail—how different parts of biomolecules exchange between functional sub-states using concerted motions. Integrative structural biology has advanced our understanding of the formation of large macromolecular complexes and how their components interact in assemblies by leveraging data from many low-resolution methods. Here, we review the growing opportunities for integrative, dynamic structural biology at the atomic scale, contending there is increasing synergistic potential between X-ray crystallography, NMR, and computer simulations to reveal a structural basis for protein conformational dynamics at high resolution.

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“…Although the chemical exchange saturation transfer (CEST) [22][23][24] experiment was previously shown to be apowerful method for characterizing the slow dynamics of GTPloaded small GTPases, [16] it failed to find the evidence of any minor conformational species in Ras·GDP in the present study (Supporting Information, Figure S1), indicating that the rate of conformational exchange of Ras·GDP falls outside the CEST-active range (20-500 s À1 ). [25] Protein conformational interconversions on the microsecond timescale could also contribute to spin relaxation (R ex ), although quantification of such fast exchange is technically more challenging.Measurement of relaxation rates of four bilinear coherences (2H z N z , 2H x N z ,2 H z N x and 2H x N x )i nt he presence of spin lock fields has previously been demonstrated as ar obust way for accurately quantifying R ex,ms ( 15 N) contributed by mstimescale motions.…”

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confidence: 99%

“…[15][16][17][18][19] Recent highpressure NMR experiments on Ras·GppNHp revealed up to three minor conformational species that were suggested as potential targets for allosteric inhibitors. [15][16][17][18][19] Recent highpressure NMR experiments on Ras·GppNHp revealed up to three minor conformational species that were suggested as potential targets for allosteric inhibitors.…”

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confidence: 99%

“…Slow conformational dynamics of Ras complexed with GTP or GTP analogues on millisecond timescales has been extensively examined in previous studies. [15][16][17][18][19] Recent highpressure NMR experiments on Ras·GppNHp revealed up to three minor conformational species that were suggested as potential targets for allosteric inhibitors. [20] In contrast to detailed characterizations of the active Ras,i nformation about the slow dynamics of the inactive Ras·GDP is scarce.…”

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Microsecond Timescale Dynamics of GDP‐Bound Ras Underlies the Formation of Novel Inhibitor‐Binding Pockets

et al. 2016

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The recent discovery of inhibitory compounds binding to distinct pockets on GDP-bound Ras has renewed the view on the druggability of this crucial cancer driver. However,t he origin of these pockets,w hicha re not readily formed in the crystal structure in the absence of the compounds,i sy et unclear.H erein, we explored the intrinsic flexibility of Ras·GDP on microsecond to millisecond timescales using relaxation-based NMR experiments,and identified substantial slow dynamics with t ex of 34 msat58 8C, whichmaps to the regions showing ah igh level of correlation with those displaying conformational differences between the inhibitorbound and free states.T hese findings,w hich have been demonstrated in both wild-type Ras and the oncogenic mutant (G12V), support the mechanism of extended conformational selection for Ras-inhibitor interactions where the small molecules redistribute the protein conformational ensemble favoring the final bound states.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under http://dx.

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A Comparative CEST NMR Study of Slow Conformational Dynamics of Small GTPases Complexed with GTP and GTP Analogues

Abstract: International audienceno abstrac

Cited by 39 publications

References 32 publications

Extending the Lifetime of Native GTP‐Bound Ras for Site‐Resolved NMR Measurements: Quantifying the Allosteric Dynamics

Extending the Lifetime of Native GTP‐Bound Ras for Site‐Resolved NMR Measurements: Quantifying the Allosteric Dynamics

Integrative, dynamic structural biology at atomic resolution—it's about time

Microsecond Timescale Dynamics of GDP‐Bound Ras Underlies the Formation of Novel Inhibitor‐Binding Pockets

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