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Probing the KRas Switch II Groove by Fluorine NMR Spectroscopy.

ABSTRACT: While there has been recent success in the development of KRas^G12C inhibitors, unmet needs for selective inhibitors of KRas^G12D and the remaining oncogenic KRas proteins remain. Here, we applied trifluoromethyl-containing ligands of KRas proteins as competitive probe ligands to assay the occupancy of the switch II pocket by ¹⁹F NMR spectroscopy. Structure-activity-relationship studies of probe ligands increased the sensitivity of the assay and identified structures that differentially detected each nucleotide state of KRas^G12D. These differences in selectivity, combined with the high resolution of ¹⁹F NMR spectroscopy, enabled this method to be expanded to assay both nucleotide states of the protein simultaneously.

SUBMITTER: Peacock DM

PROVIDER: S-EPMC9594042 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Probing the KRas Switch II Groove by Fluorine NMR Spectroscopy.

Peacock D Matthew DM Kelly Mark J S MJS Shokat Kevan M KM

ACS chemical biology 20220927 10

While there has been recent success in the development of KRasG12C inhibitors, unmet needs for selective inhibitors of KRasG12D and the remaining oncogenic KRas proteins remain. Here, we applied trifluoromethyl-containing ligands of KRas proteins as competitive probe ligands to assay the occupancy of the switch II pocket by 19F NMR spectroscopy. Structure-activity-relationship studies of probe ligands increased the sensitivity of the assay and identified structur ...[more]

PMID: 36166818

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Project description:Post-translational modification (PTM) of proteins is of critical importance to the regulation of many cellular processes in eukaryotic organisms. One of the most well-studied protein PTMs is methylation, wherein an enzyme catalyzes the transfer of a methyl group from a cofactor to a lysine or arginine side chain. Lysine methylation is especially abundant in the histone tails and is an important marker for denoting active or repressed genes. Given their relevance to transcriptional regulation, the study of methyltransferase function through in vitro experiments is an important stepping stone toward understanding the complex mechanisms of regulated gene expression. To date, most methyltransferase characterization strategies rely on the use of radioactive cofactors, detection of a methyl transfer byproduct, or discontinuous-type assays. Although such methods are suitable for some applications, information about multiple methylation events and kinetic intermediates is often lost. Herein, we describe the use of two-dimensional NMR to monitor mono-, di-, and trimethylation in a single reaction tube. To do so, we incorporated 13C into the donor methyl group of the enzyme cofactor S-adenosyl methionine. In this way, we may study enzymatic methylation by monitoring the appearance of distinct resonances corresponding to mono-, di-, or trimethyl lysine without the need to isotopically enrich the substrate. To demonstrate the capabilities of this method, we evaluated the activity of three lysine methyltransferases, Set7, MWRAD2 (MLL1 complex), and PRDM9, toward the histone H3 tail. We monitored mono- or multimethylation of histone H3 tail at lysine 4 through sequential short two-dimensional heteronuclear single quantum coherence experiments and fit the resulting progress curves to first-order kinetic models. In summary, NMR detection of PTMs in one-pot, real-time reaction using facile cofactor isotopic enrichment shows promise as a method toward understanding the intricate mechanisms of methyltransferases and other enzymes.

Dataset Information

Probing the KRas Switch II Groove by Fluorine NMR Spectroscopy.

Publications

Probing the KRas Switch II Groove by Fluorine NMR Spectroscopy.

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