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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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Conformational transition from inactive to active Srcmonitoredby NMR. (a,b) Structures of (a) inactive Src (2SRC) and (b) activeSrc (1Y57) showing Lys295 and Lys315 in the N-lobe of the kinase domain.Glu310 and Trp260 are also shown, as are portions of the SH3 and SH2domains on the “backside” of the Src kinase domain.(c) SH3 peptide ligand (NAc-VSLARRPLPPLP-NH2) was titratedinto 135 μM [13C]-methylated Src SH3SH2KD, and [1H,13C] HSQC spectra were acquired at each titrationpoint. The left-most spectrum is [13C]-methylated Src SH3SH2KDwith no ligand (bold outline), and the following six spectra containincreasing molar equivalents of peptide ligand. Boxes throughout panelsc and d show the resonance frequencies of Lys295 and Lys315 in theabsence of ligand. Peak assignments are indicated in the first spectrum,and the asterisk indicates an unidentified peak. (d) SH2 peptide ligand(Caffeic acid-pYEEIE) titrated into the [13C]-methylatedSrc SH3SH2KD sample containing 10 molar equiv of SH3 peptide ligand(last panel in part c). The bolded spectrum is Src SH3SH2KD proteinin the presence of saturating amounts of both SH3 and SH2 domain ligands.The final panel shows the spectrum acquired after dialysis of theligand-saturated Src SH3SH2KD sample. (e) (left) [1H,13C] HSQC spectrum of the [13C]-methylated Src kinasedomain (Src KD). (right) Superposition of the Src KD spectrum (blue)with the [1H,13C] HSQC spectrum of SH3SH2KDafter saturation with both SH3 and SH2 ligands (black).
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fig3: Conformational transition from inactive to active Srcmonitoredby NMR. (a,b) Structures of (a) inactive Src (2SRC) and (b) activeSrc (1Y57) showing Lys295 and Lys315 in the N-lobe of the kinase domain.Glu310 and Trp260 are also shown, as are portions of the SH3 and SH2domains on the “backside” of the Src kinase domain.(c) SH3 peptide ligand (NAc-VSLARRPLPPLP-NH2) was titratedinto 135 μM [13C]-methylated Src SH3SH2KD, and [1H,13C] HSQC spectra were acquired at each titrationpoint. The left-most spectrum is [13C]-methylated Src SH3SH2KDwith no ligand (bold outline), and the following six spectra containincreasing molar equivalents of peptide ligand. Boxes throughout panelsc and d show the resonance frequencies of Lys295 and Lys315 in theabsence of ligand. Peak assignments are indicated in the first spectrum,and the asterisk indicates an unidentified peak. (d) SH2 peptide ligand(Caffeic acid-pYEEIE) titrated into the [13C]-methylatedSrc SH3SH2KD sample containing 10 molar equiv of SH3 peptide ligand(last panel in part c). The bolded spectrum is Src SH3SH2KD proteinin the presence of saturating amounts of both SH3 and SH2 domain ligands.The final panel shows the spectrum acquired after dialysis of theligand-saturated Src SH3SH2KD sample. (e) (left) [1H,13C] HSQC spectrum of the [13C]-methylated Src kinasedomain (Src KD). (right) Superposition of the Src KD spectrum (blue)with the [1H,13C] HSQC spectrum of SH3SH2KDafter saturation with both SH3 and SH2 ligands (black).

Mentions: The resolvedmethyl resonances of Lys295 and 315 provide two separateprobes within the Src SH3SH2KD protein to monitor conformational changesduring the course of Src activation. Lys315 is one and a half turnsaway from Glu310 on the C-helix and in the autoinhibited Src structureprojects away from the N-lobe of the kinase domain toward the linkerbetween SH2 and kinase domains, making extensive contacts with Trp260(Figure 3a). Src activation leads to a largeshift in the position of the SH3/SH2 domains and the SH2-kinase linkerregion (Figure 3b). Peptide ligands that targetthe Src SH3 and/or SH2 domains compete with the autoinhibited formand activate the Src kinase.28 We titratedtwo peptides, VSLARRPLPPLP and pYEEIE (ligands for the Src SH3 andSH2 domains, respectively), into the NMR sample containing [13C]-methylated Src SH3SH2KD (Figure 3c,d).Addition of increasing concentration of SH3 ligand causes spectralchanges; specifically, the peak corresponding to Lys295 disappearsover the course of SH3 ligand titration, due to line broadening and/orchemical shift change that results in overlap with the neighboringpeak (Figure 3c). The Lys315 methyl peak exhibitsslow exchange behavior as SH3 ligand concentration increases (Figure 3c). Saturation with SH3 peptide ligand results inthe emergence of a new peak at a 1H frequency of 2.28 ppmand complete loss of the original Lys315 signal present in the spectrumof free Src SH3SH2KD.


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

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

Conformational transition from inactive to active Srcmonitoredby NMR. (a,b) Structures of (a) inactive Src (2SRC) and (b) activeSrc (1Y57) showing Lys295 and Lys315 in the N-lobe of the kinase domain.Glu310 and Trp260 are also shown, as are portions of the SH3 and SH2domains on the “backside” of the Src kinase domain.(c) SH3 peptide ligand (NAc-VSLARRPLPPLP-NH2) was titratedinto 135 μM [13C]-methylated Src SH3SH2KD, and [1H,13C] HSQC spectra were acquired at each titrationpoint. The left-most spectrum is [13C]-methylated Src SH3SH2KDwith no ligand (bold outline), and the following six spectra containincreasing molar equivalents of peptide ligand. Boxes throughout panelsc and d show the resonance frequencies of Lys295 and Lys315 in theabsence of ligand. Peak assignments are indicated in the first spectrum,and the asterisk indicates an unidentified peak. (d) SH2 peptide ligand(Caffeic acid-pYEEIE) titrated into the [13C]-methylatedSrc SH3SH2KD sample containing 10 molar equiv of SH3 peptide ligand(last panel in part c). The bolded spectrum is Src SH3SH2KD proteinin the presence of saturating amounts of both SH3 and SH2 domain ligands.The final panel shows the spectrum acquired after dialysis of theligand-saturated Src SH3SH2KD sample. (e) (left) [1H,13C] HSQC spectrum of the [13C]-methylated Src kinasedomain (Src KD). (right) Superposition of the Src KD spectrum (blue)with the [1H,13C] HSQC spectrum of SH3SH2KDafter saturation with both SH3 and SH2 ligands (black).
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fig3: Conformational transition from inactive to active Srcmonitoredby NMR. (a,b) Structures of (a) inactive Src (2SRC) and (b) activeSrc (1Y57) showing Lys295 and Lys315 in the N-lobe of the kinase domain.Glu310 and Trp260 are also shown, as are portions of the SH3 and SH2domains on the “backside” of the Src kinase domain.(c) SH3 peptide ligand (NAc-VSLARRPLPPLP-NH2) was titratedinto 135 μM [13C]-methylated Src SH3SH2KD, and [1H,13C] HSQC spectra were acquired at each titrationpoint. The left-most spectrum is [13C]-methylated Src SH3SH2KDwith no ligand (bold outline), and the following six spectra containincreasing molar equivalents of peptide ligand. Boxes throughout panelsc and d show the resonance frequencies of Lys295 and Lys315 in theabsence of ligand. Peak assignments are indicated in the first spectrum,and the asterisk indicates an unidentified peak. (d) SH2 peptide ligand(Caffeic acid-pYEEIE) titrated into the [13C]-methylatedSrc SH3SH2KD sample containing 10 molar equiv of SH3 peptide ligand(last panel in part c). The bolded spectrum is Src SH3SH2KD proteinin the presence of saturating amounts of both SH3 and SH2 domain ligands.The final panel shows the spectrum acquired after dialysis of theligand-saturated Src SH3SH2KD sample. (e) (left) [1H,13C] HSQC spectrum of the [13C]-methylated Src kinasedomain (Src KD). (right) Superposition of the Src KD spectrum (blue)with the [1H,13C] HSQC spectrum of SH3SH2KDafter saturation with both SH3 and SH2 ligands (black).
Mentions: The resolvedmethyl resonances of Lys295 and 315 provide two separateprobes within the Src SH3SH2KD protein to monitor conformational changesduring the course of Src activation. Lys315 is one and a half turnsaway from Glu310 on the C-helix and in the autoinhibited Src structureprojects away from the N-lobe of the kinase domain toward the linkerbetween SH2 and kinase domains, making extensive contacts with Trp260(Figure 3a). Src activation leads to a largeshift in the position of the SH3/SH2 domains and the SH2-kinase linkerregion (Figure 3b). Peptide ligands that targetthe Src SH3 and/or SH2 domains compete with the autoinhibited formand activate the Src kinase.28 We titratedtwo peptides, VSLARRPLPPLP and pYEEIE (ligands for the Src SH3 andSH2 domains, respectively), into the NMR sample containing [13C]-methylated Src SH3SH2KD (Figure 3c,d).Addition of increasing concentration of SH3 ligand causes spectralchanges; specifically, the peak corresponding to Lys295 disappearsover the course of SH3 ligand titration, due to line broadening and/orchemical shift change that results in overlap with the neighboringpeak (Figure 3c). The Lys315 methyl peak exhibitsslow exchange behavior as SH3 ligand concentration increases (Figure 3c). Saturation with SH3 peptide ligand results inthe emergence of a new peak at a 1H frequency of 2.28 ppmand complete loss of the original Lys315 signal present in the spectrumof free Src SH3SH2KD.

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