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

Chen, Xiaomin; Yao, Haijie; Wang, Hui; Mao, Yunyun; Liú, Dan; Long, Dong

doi:10.1002/ange.201812902

Cited by 3 publications

(3 citation statements)

References 39 publications

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“…For GTP-bound WT K-Ras and the G12C and G12D mutants, around 80% of the backbone resonances were assigned recently, but the entire Switch II and a substantial number of resonances of Switch I were still missing 11 . As multidimensional NMR applications of Ras when bound to native GTP are impeded by the real-time hydrolysis of GTP, the addition of GEF was found to significantly extend the lifetime of H-Ras, allowing dynamics measurements of a larger number of residues, including several residues of the Switch regions 12 . A subsequent combined X-ray crystallography and one-dimensional 1 H solution NMR study of WT K-Ras bound to GppCH 2 p found a significantly increased state 1 population compared with that of H-Ras, whereas the K-Ras G12D mutant favored state 2 (ref.…”

Section: Mainmentioning

confidence: 99%

Excited-state observation of active K-Ras reveals differential structural dynamics of wild-type versus oncogenic G12D and G12C mutants

Hansen,

Xiang,

Yuan

et al. 2023

Nat Struct Mol Biol

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Despite the prominent role of the K-Ras protein in many different types of human cancer, major gaps in atomic-level information severely limit our understanding of its functions in health and disease. Here, we report the quantitative backbone structural dynamics of K-Ras by solution nuclear magnetic resonance spectroscopy of the active state of wild-type K-Ras bound to guanosine triphosphate (GTP) nucleotide and two of its oncogenic P-loop mutants, G12D and G12C, using a new nanoparticle-assisted spin relaxation method, relaxation dispersion and chemical exchange saturation transfer experiments covering the entire range of timescales from picoseconds to milliseconds. Our combined experiments allow detection and analysis of the functionally critical Switch I and Switch II regions, which have previously remained largely unobservable by X-ray crystallography and nuclear magnetic resonance spectroscopy. Our data reveal cooperative transitions of K-Ras·GTP to a highly dynamic excited state that closely resembles the partially disordered K-Ras·GDP state. These results advance our understanding of differential GTPase activities and signaling properties of the wild type versus mutants and may thus guide new strategies for the development of therapeutics.

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Section: Mainmentioning

confidence: 99%

Excited-state observation of active K-Ras reveals differential structural dynamics of wild-type versus oncogenic G12D and G12C mutants

Hansen,

Xiang,

Yuan

et al. 2023

Nat Struct Mol Biol

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“…S2 ). This observation provides further evidence for the vital role of the enzyme conformational dynamics in the enhancement of enzyme catalysis 35 , 36 , 37 , 38 , through stabilization of the catalytically competent conformation of the enzyme–substrate complex exerted by MDL-801.…”

Section: Resultsmentioning

confidence: 55%

Mechanism of allosteric activation of SIRT6 revealed by the action of rationally designed activators

Chen

Wei

et al. 2021

Acta Pharmaceutica Sinica B

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The recent discovery of activator compounds binding to an allosteric site on the NAD + -dependent protein lysine deacetylase, sirtuin 6 (SIRT6) has attracted interest and presents a pharmaceutical target for aging-related and cancer diseases. However, the mechanism underlying allosteric activation of SIRT6 by the activator MDL-801 remains largely elusive because no major conformational changes are observed upon activator binding. By combining molecular dynamics simulations with biochemical and kinetic analyses of wild-type SIRT6 and its variant M136A, we show that conformational rotation of 2-methyl-4-fluoro-5-bromo substituent on the right phenyl ring (R-ring) of MDL-801, which uncovers previously unseen hydrophobic interactions, contributes to increased activating deacetylation activity of SIRT6. This hypothesis is further supported by the two newly synthesized MDL-801 derivatives through the removal of the 5-Br atom on the R-ring (MDL-801-D1) or the restraint of the rotation of the R-ring (MDL-801-D2). We further propose that the 5-Br atom serves as an allosteric driver that controls the ligand allosteric efficacy. Our study highlights the effect of allosteric enzyme catalytic activity by activator binding and provides a rational approach for enhancing deacetylation activity.

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“…Both the active and inactive forms of Ras, however, are inherently flexible, populating rare conformers distinct from the native structures and presenting alternative opportunities for drug discovery (8)(9)(10)(11). For example, in GTP-bound active Ras, a major and minor state (termed states 2 and 1, respectively) coexist in solution and exchange on a millisecond timescale, with state 1 showing surface roughness unobserved in the major state (12)(13)(14)(15)(16)(17)(18). The direct visibility of state 1 in the one-dimensional 31 P NMR spectra of active Ras largely facilitated its early discovery and characterization (12,19).…”

mentioning

confidence: 99%

Unveiling the “invisible” druggable conformations of GDP-bound inactive Ras

Liu

Mao

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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The prevalent view on whether Ras is druggable has gradually changed in the recent decade with the discovery of effective inhibitors binding to cryptic sites unseen in the native structures. Despite the promising advances, therapeutics development toward higher potency and specificity is challenged by the elusive nature of these binding pockets. Here we derive a conformational ensemble of guanosine diphosphate (GDP)-bound inactive Ras by integrating spin relaxation-validated atomistic simulation with NMR chemical shifts and residual dipolar couplings, which provides a quantitative delineation of the intrinsic dynamics up to the microsecond timescale. The experimentally informed ensemble unequivocally demonstrates the preformation of both surface-exposed and buried cryptic sites in Ras•GDP, advocating design of inhibition by targeting the transient druggable conformers that are invisible to conventional experimental methods. The viability of the ensemble-based rational design has been established by retrospective testing of the ability of the Ras•GDP ensemble to identify known ligands from decoys in virtual screening.

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

Cited by 3 publications

References 39 publications

Excited-state observation of active K-Ras reveals differential structural dynamics of wild-type versus oncogenic G12D and G12C mutants

Excited-state observation of active K-Ras reveals differential structural dynamics of wild-type versus oncogenic G12D and G12C mutants

Mechanism of allosteric activation of SIRT6 revealed by the action of rationally designed activators

Unveiling the “invisible” druggable conformations of GDP-bound inactive Ras

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