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A selective NMR probe to monitor the conformational transition from inactive to active kinase.

Xie Q, Fulton DB, Andreotti AH - ACS Chem. Biol. (2014)

Bottom Line: Monitoring the conformational changes that drive activation and inactivation of the catalytic kinase core is a challenging experimental problem due to the dynamic nature of these enzymes.We apply [(13)C] reductive methylation to chemically introduce NMR-active nuclei into unlabeled protein kinases.This approach provides a solution based method to complement X-ray crystallographic data and can be applied to nearly any kinase, regardless of size or method of production.

View Article: PubMed Central - PubMed

Affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University , Ames, Iowa 50011, United States.

ABSTRACT
Kinases control many aspects of cellular signaling and are therefore therapeutic targets for numerous disease states. Monitoring the conformational changes that drive activation and inactivation of the catalytic kinase core is a challenging experimental problem due to the dynamic nature of these enzymes. We apply [(13)C] reductive methylation to chemically introduce NMR-active nuclei into unlabeled protein kinases. The results demonstrate that solution NMR spectroscopy can be used to monitor specific changes in the chemical environment of structurally important lysines in a [(13)C]-methylated kinase as it shifts from the inactive to active state. This approach provides a solution based method to complement X-ray crystallographic data and can be applied to nearly any kinase, regardless of size or method of production.

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Extension of method toadditional kinases. (a,b) Dimethyl lysineregion of spectra acquired for [13C]-methylated Btk (a)and Csk (kinase domains and SH3-SH2-kinase domain constructs) (b).Non-degenerate resonances are indicated with a dashed box. (c) Btkkinase domain drug titrations. A 100 μM sample of [13C]-methylated Btk kinase domain (Tyr551 is mutated to Glu to correspondto protein used in the reported crystal structure35) was titrated with either B43 (top row) or dasatinib (bottomrow) at the indicated molar equivalents. [1H,13C] HSQC spectra shown are acquired before addition of drug or ateach titration point. The Btk kinase domain also carries the Y617Pmutation that facilitates bacterial expression.39
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fig4: Extension of method toadditional kinases. (a,b) Dimethyl lysineregion of spectra acquired for [13C]-methylated Btk (a)and Csk (kinase domains and SH3-SH2-kinase domain constructs) (b).Non-degenerate resonances are indicated with a dashed box. (c) Btkkinase domain drug titrations. A 100 μM sample of [13C]-methylated Btk kinase domain (Tyr551 is mutated to Glu to correspondto protein used in the reported crystal structure35) was titrated with either B43 (top row) or dasatinib (bottomrow) at the indicated molar equivalents. [1H,13C] HSQC spectra shown are acquired before addition of drug or ateach titration point. The Btk kinase domain also carries the Y617Pmutation that facilitates bacterial expression.39

Mentions: The proof-of-principle experimentin Figure 3 demonstrates that NMR spectroscopy,using [13C]-methylatedlysine side chains as probes, reports on the conformational ensembleof the Src kinase domain under a range of conditions. In light ofthe inconvenient exchange regime/chemical shift degeneracy of theLys295 resonance, the well-resolved Src Lys315 peak provides a convenient,alternative signal that monitors the complete conformational transitionbetween inactive and active Src (Figure 3c,d).While Src Lys315 is not strictly conserved across kinase families,we wondered whether other kinases would similarly exhibit multipleresolved dimethyl lysine resonances that, in the event of unfavorablespectral properties for the conserved β3 lysine, could be usedto monitor the complete conformational transition between inactiveand active kinase. To this end, we expressed and purified two additionalkinases, Btk and Csk, and subjected these proteins to reductive methylation.Acquisition of [1H,13C] HSQC spectra for methylatedBtk and Csk (Figure 4a,b) shows that thesekinases, like Src, exhibit multiple nondegenerate dimethyl resonances.Moreover, a comparison of the isolated Btk and Csk kinase domainsto their corresponding multidomain proteins (SH3SH2KD) shows spectraldifferences in the region of the resolved dimethyl lysine resonances(Figure 4a,b) consistent with activity differences,and hence conformational differences, in the kinase domain inducedby the presence of the non-catalytic domains.33,34 While residue-specific assignments and characterization of thesesystems are beyond the scope of the current work, these data suggestthat the presence of multiple spectroscopic probes may be a generalfeature of reductively methylated kinases.


A selective NMR probe to monitor the conformational transition from inactive to active kinase.

Xie Q, Fulton DB, Andreotti AH - ACS Chem. Biol. (2014)

Extension of method toadditional kinases. (a,b) Dimethyl lysineregion of spectra acquired for [13C]-methylated Btk (a)and Csk (kinase domains and SH3-SH2-kinase domain constructs) (b).Non-degenerate resonances are indicated with a dashed box. (c) Btkkinase domain drug titrations. A 100 μM sample of [13C]-methylated Btk kinase domain (Tyr551 is mutated to Glu to correspondto protein used in the reported crystal structure35) was titrated with either B43 (top row) or dasatinib (bottomrow) at the indicated molar equivalents. [1H,13C] HSQC spectra shown are acquired before addition of drug or ateach titration point. The Btk kinase domain also carries the Y617Pmutation that facilitates bacterial expression.39
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4301085&req=5

fig4: Extension of method toadditional kinases. (a,b) Dimethyl lysineregion of spectra acquired for [13C]-methylated Btk (a)and Csk (kinase domains and SH3-SH2-kinase domain constructs) (b).Non-degenerate resonances are indicated with a dashed box. (c) Btkkinase domain drug titrations. A 100 μM sample of [13C]-methylated Btk kinase domain (Tyr551 is mutated to Glu to correspondto protein used in the reported crystal structure35) was titrated with either B43 (top row) or dasatinib (bottomrow) at the indicated molar equivalents. [1H,13C] HSQC spectra shown are acquired before addition of drug or ateach titration point. The Btk kinase domain also carries the Y617Pmutation that facilitates bacterial expression.39
Mentions: The proof-of-principle experimentin Figure 3 demonstrates that NMR spectroscopy,using [13C]-methylatedlysine side chains as probes, reports on the conformational ensembleof the Src kinase domain under a range of conditions. In light ofthe inconvenient exchange regime/chemical shift degeneracy of theLys295 resonance, the well-resolved Src Lys315 peak provides a convenient,alternative signal that monitors the complete conformational transitionbetween inactive and active Src (Figure 3c,d).While Src Lys315 is not strictly conserved across kinase families,we wondered whether other kinases would similarly exhibit multipleresolved dimethyl lysine resonances that, in the event of unfavorablespectral properties for the conserved β3 lysine, could be usedto monitor the complete conformational transition between inactiveand active kinase. To this end, we expressed and purified two additionalkinases, Btk and Csk, and subjected these proteins to reductive methylation.Acquisition of [1H,13C] HSQC spectra for methylatedBtk and Csk (Figure 4a,b) shows that thesekinases, like Src, exhibit multiple nondegenerate dimethyl resonances.Moreover, a comparison of the isolated Btk and Csk kinase domainsto their corresponding multidomain proteins (SH3SH2KD) shows spectraldifferences in the region of the resolved dimethyl lysine resonances(Figure 4a,b) consistent with activity differences,and hence conformational differences, in the kinase domain inducedby the presence of the non-catalytic domains.33,34 While residue-specific assignments and characterization of thesesystems are beyond the scope of the current work, these data suggestthat the presence of multiple spectroscopic probes may be a generalfeature of reductively methylated kinases.

Bottom Line: Monitoring the conformational changes that drive activation and inactivation of the catalytic kinase core is a challenging experimental problem due to the dynamic nature of these enzymes.We apply [(13)C] reductive methylation to chemically introduce NMR-active nuclei into unlabeled protein kinases.This approach provides a solution based method to complement X-ray crystallographic data and can be applied to nearly any kinase, regardless of size or method of production.

View Article: PubMed Central - PubMed

Affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University , Ames, Iowa 50011, United States.

ABSTRACT
Kinases control many aspects of cellular signaling and are therefore therapeutic targets for numerous disease states. Monitoring the conformational changes that drive activation and inactivation of the catalytic kinase core is a challenging experimental problem due to the dynamic nature of these enzymes. We apply [(13)C] reductive methylation to chemically introduce NMR-active nuclei into unlabeled protein kinases. The results demonstrate that solution NMR spectroscopy can be used to monitor specific changes in the chemical environment of structurally important lysines in a [(13)C]-methylated kinase as it shifts from the inactive to active state. This approach provides a solution based method to complement X-ray crystallographic data and can be applied to nearly any kinase, regardless of size or method of production.

Show MeSH