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Engineered allosteric activation of kinases in living cells.

Karginov AV, Ding F, Kota P, Dokholyan NV, Hahn KM - Nat. Biotechnol. (2010)

Bottom Line: Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems.A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity but does not affect other protein functions (Fig. 1a).Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

ABSTRACT
Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. Here we describe an approach to confer allosteric regulation specifically on the catalytic activity of protein kinases. A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity but does not affect other protein functions (Fig. 1a). Catalytic activity is restored by addition of rapamycin or non-immunosuppresive rapamycin analogs. Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain. Drug binding restores activity by increasing rigidity. We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells and show that FAK is involved in the regulation of membrane dynamics. Successful regulation of Src and p38 by insertion of the rapamycin-responsive element at the same conserved site used in FAK suggests that our strategy will be applicable to other kinases.

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Mechanism of regulation by iFKBP; Src regulation. (A) The portion of the FAK catalytic domain targeted for insertion of iFKBP (blue) and the G-loop (red). (B) Dynamic correlation analysis of the wild type FAK catalytic domain (red, positive correlation; blue, negative correlation). The circled region indicates strong negative correlation between the movement of the insertion loop and the G-loop. (C) Tube representation depicting changes in the dynamics of the FAK catalytic domain’s N-terminal lobe, based on molecular dynamics simulations. Warmer colors and thicker backbone correspond to higher RMSF values, reflecting the degree of free movement within the structure. The red arrows points to the G-loop. (D) Root mean square fluctuation (RMSF) of amino acids in FAK and RapR-FAK(arrow indicates G-loop). The break in the wild type FAK graph corresponds to the iFKBP insert in RapR-FAK. (E) Regulation of Src kinase by insertion of iFKBP. HEK293T cells co-expressing the indicated myc-tagged Src construct and GFP-FRB were treated with either 200 nMrapamycin or ethanol solvent control. The kinase activity of immunoprecipitatedSrc was tested as in 2A.
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Figure 4: Mechanism of regulation by iFKBP; Src regulation. (A) The portion of the FAK catalytic domain targeted for insertion of iFKBP (blue) and the G-loop (red). (B) Dynamic correlation analysis of the wild type FAK catalytic domain (red, positive correlation; blue, negative correlation). The circled region indicates strong negative correlation between the movement of the insertion loop and the G-loop. (C) Tube representation depicting changes in the dynamics of the FAK catalytic domain’s N-terminal lobe, based on molecular dynamics simulations. Warmer colors and thicker backbone correspond to higher RMSF values, reflecting the degree of free movement within the structure. The red arrows points to the G-loop. (D) Root mean square fluctuation (RMSF) of amino acids in FAK and RapR-FAK(arrow indicates G-loop). The break in the wild type FAK graph corresponds to the iFKBP insert in RapR-FAK. (E) Regulation of Src kinase by insertion of iFKBP. HEK293T cells co-expressing the indicated myc-tagged Src construct and GFP-FRB were treated with either 200 nMrapamycin or ethanol solvent control. The kinase activity of immunoprecipitatedSrc was tested as in 2A.

Mentions: In order to understand the molecular mechanism of RapR-FAK allosteric regulation and explore the generalizability of the approach, we performed molecular dynamics simulations24, 25. Combined with the biochemical data, the computational analysis indicated a mechanism for iFKBP-mediated regulation. The point where iFKBP is inserted connects via a β strand to FAK’s Gly loop (G-loop), a structural feature critical for positioning the ATP phosphate groups in the catalytic site (Fig 4A) 26. Molecular dynamics analysis indicated that the conformational mobility of the G-loop is correlated with that of the FAK region where iFKBP is inserted (the ‘insertion loop’, Fig. 4B), suggesting that the dynamics of the insertion loop could affect the dynamics of the G-loop and thereby change the catalytic activity. Comparison of wt FAK and RapR-FAK dynamics indicated that the amplitude of G-loop conformational dynamics is dramatically increased when iFKBP is inserted in the catalytic domain. These dynamics decreased back to wild-type levels upon binding to rapamycin/FRB (Fig. 4C, D, supplementary movie S3). Based on this analysis, we postulate that the effectiveness of the G-loop in the phosphate transfer reaction is reduced due to greater conformational flexibility produced by insertion of iFKBP. Interaction with rapamycin and FRB stabilizes the G loop to rescue FAK catalytic activity. Molecular dynamics analysis was consistent with empirical measurements; dynamics analysis of the FAK-iFKBP445 variant suggested that its longer linkers decreased coupling between the iFKBP insert and G-loop dynamics (Supplementary Fig. S13), resulting in the less effective FAK inhibition observed in biochemical studies (Fig. 2A, FAK-iFKBP445 construct). In contrast, insertion of iFKBP without any linkers restricted the structural dynamics of iFKBP, consistent with the observed minimal effects on catalytic activity (Supplementary Fig. S13, FAK-iFKBP442-8 construct). In summary, computational analysis indicates that the allosteric modulation of RapR-FAK activity results from dynamic coupling of the optimized iFKBP insertion and the kinase G-loop, highly conserved structural features in all known kinases 26.


Engineered allosteric activation of kinases in living cells.

Karginov AV, Ding F, Kota P, Dokholyan NV, Hahn KM - Nat. Biotechnol. (2010)

Mechanism of regulation by iFKBP; Src regulation. (A) The portion of the FAK catalytic domain targeted for insertion of iFKBP (blue) and the G-loop (red). (B) Dynamic correlation analysis of the wild type FAK catalytic domain (red, positive correlation; blue, negative correlation). The circled region indicates strong negative correlation between the movement of the insertion loop and the G-loop. (C) Tube representation depicting changes in the dynamics of the FAK catalytic domain’s N-terminal lobe, based on molecular dynamics simulations. Warmer colors and thicker backbone correspond to higher RMSF values, reflecting the degree of free movement within the structure. The red arrows points to the G-loop. (D) Root mean square fluctuation (RMSF) of amino acids in FAK and RapR-FAK(arrow indicates G-loop). The break in the wild type FAK graph corresponds to the iFKBP insert in RapR-FAK. (E) Regulation of Src kinase by insertion of iFKBP. HEK293T cells co-expressing the indicated myc-tagged Src construct and GFP-FRB were treated with either 200 nMrapamycin or ethanol solvent control. The kinase activity of immunoprecipitatedSrc was tested as in 2A.
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Related In: Results  -  Collection

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Figure 4: Mechanism of regulation by iFKBP; Src regulation. (A) The portion of the FAK catalytic domain targeted for insertion of iFKBP (blue) and the G-loop (red). (B) Dynamic correlation analysis of the wild type FAK catalytic domain (red, positive correlation; blue, negative correlation). The circled region indicates strong negative correlation between the movement of the insertion loop and the G-loop. (C) Tube representation depicting changes in the dynamics of the FAK catalytic domain’s N-terminal lobe, based on molecular dynamics simulations. Warmer colors and thicker backbone correspond to higher RMSF values, reflecting the degree of free movement within the structure. The red arrows points to the G-loop. (D) Root mean square fluctuation (RMSF) of amino acids in FAK and RapR-FAK(arrow indicates G-loop). The break in the wild type FAK graph corresponds to the iFKBP insert in RapR-FAK. (E) Regulation of Src kinase by insertion of iFKBP. HEK293T cells co-expressing the indicated myc-tagged Src construct and GFP-FRB were treated with either 200 nMrapamycin or ethanol solvent control. The kinase activity of immunoprecipitatedSrc was tested as in 2A.
Mentions: In order to understand the molecular mechanism of RapR-FAK allosteric regulation and explore the generalizability of the approach, we performed molecular dynamics simulations24, 25. Combined with the biochemical data, the computational analysis indicated a mechanism for iFKBP-mediated regulation. The point where iFKBP is inserted connects via a β strand to FAK’s Gly loop (G-loop), a structural feature critical for positioning the ATP phosphate groups in the catalytic site (Fig 4A) 26. Molecular dynamics analysis indicated that the conformational mobility of the G-loop is correlated with that of the FAK region where iFKBP is inserted (the ‘insertion loop’, Fig. 4B), suggesting that the dynamics of the insertion loop could affect the dynamics of the G-loop and thereby change the catalytic activity. Comparison of wt FAK and RapR-FAK dynamics indicated that the amplitude of G-loop conformational dynamics is dramatically increased when iFKBP is inserted in the catalytic domain. These dynamics decreased back to wild-type levels upon binding to rapamycin/FRB (Fig. 4C, D, supplementary movie S3). Based on this analysis, we postulate that the effectiveness of the G-loop in the phosphate transfer reaction is reduced due to greater conformational flexibility produced by insertion of iFKBP. Interaction with rapamycin and FRB stabilizes the G loop to rescue FAK catalytic activity. Molecular dynamics analysis was consistent with empirical measurements; dynamics analysis of the FAK-iFKBP445 variant suggested that its longer linkers decreased coupling between the iFKBP insert and G-loop dynamics (Supplementary Fig. S13), resulting in the less effective FAK inhibition observed in biochemical studies (Fig. 2A, FAK-iFKBP445 construct). In contrast, insertion of iFKBP without any linkers restricted the structural dynamics of iFKBP, consistent with the observed minimal effects on catalytic activity (Supplementary Fig. S13, FAK-iFKBP442-8 construct). In summary, computational analysis indicates that the allosteric modulation of RapR-FAK activity results from dynamic coupling of the optimized iFKBP insertion and the kinase G-loop, highly conserved structural features in all known kinases 26.

Bottom Line: Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems.A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity but does not affect other protein functions (Fig. 1a).Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

ABSTRACT
Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. Here we describe an approach to confer allosteric regulation specifically on the catalytic activity of protein kinases. A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity but does not affect other protein functions (Fig. 1a). Catalytic activity is restored by addition of rapamycin or non-immunosuppresive rapamycin analogs. Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain. Drug binding restores activity by increasing rigidity. We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells and show that FAK is involved in the regulation of membrane dynamics. Successful regulation of Src and p38 by insertion of the rapamycin-responsive element at the same conserved site used in FAK suggests that our strategy will be applicable to other kinases.

Show MeSH
Related in: MedlinePlus