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Biochemical and biophysical characterization of four EphB kinase domains reveals contrasting thermodynamic, kinetic and inhibition profiles.

Overman RC, Debreczeni JE, Truman CM, McAlister MS, Attwood TK - Biosci. Rep. (2013)

Bottom Line: A greater understanding of the similarities and differences within this small, highly conserved family of tyrosine kinases will be essential to the identification of effective therapeutic opportunities for disease intervention.Our findings have led us to speculate about both their biological significance and potential routes for generating EphB isozyme-selective small-molecule inhibitors.Our comprehensive methodologies provide a template for similar in-depth studies of other kinase superfamily members.

View Article: PubMed Central - PubMed

Affiliation: AstraZeneca PLC, Alderley Park, Cheshire, SK10 4TG, UK. ross.overman@astrazeneca.com

ABSTRACT
The Eph (erythropoietin-producing hepatocellular carcinoma) B receptors are important in a variety of cellular processes through their roles in cell-to-cell contact and signalling; their up-regulation and down-regulation has been shown to have implications in a variety of cancers. A greater understanding of the similarities and differences within this small, highly conserved family of tyrosine kinases will be essential to the identification of effective therapeutic opportunities for disease intervention. In this study, we have developed a route to production of multi-milligram quantities of highly purified, homogeneous, recombinant protein for the kinase domain of these human receptors in Escherichia coli. Analyses of these isolated catalytic fragments have revealed stark contrasts in their amenability to recombinant expression and their physical properties: e.g., a >16°C variance in thermal stability, a 3-fold difference in catalytic activity and disparities in their inhibitor binding profiles. We find EphB3 to be an outlier in terms of both its intrinsic stability, and more importantly its ligand-binding properties. Our findings have led us to speculate about both their biological significance and potential routes for generating EphB isozyme-selective small-molecule inhibitors. Our comprehensive methodologies provide a template for similar in-depth studies of other kinase superfamily members.

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Related in: MedlinePlus

Chaotrope-induced denaturation of EphB kinaseUnfolding EphB kinases in the presence of increasing GdnHCl was monitored by following the progressive quenching of internal tryptophan fluorescence (excitation 295 nm, emission 345 nm), as described. The y-axis shows relative fluorescence units measured at 345 nm averaged from five scans of three independent experiments. Curves were fitted to both two-state (broken line) and three-state (unbroken line) models [28,29], using Prism software (GraphPad Software, Inc.).
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Figure 3: Chaotrope-induced denaturation of EphB kinaseUnfolding EphB kinases in the presence of increasing GdnHCl was monitored by following the progressive quenching of internal tryptophan fluorescence (excitation 295 nm, emission 345 nm), as described. The y-axis shows relative fluorescence units measured at 345 nm averaged from five scans of three independent experiments. Curves were fitted to both two-state (broken line) and three-state (unbroken line) models [28,29], using Prism software (GraphPad Software, Inc.).

Mentions: In an attempt to follow the unfolding events of each of the four kinases in greater detail, chaotrope-induced unfolding was performed. Internal tryptophan fluorescence of the four kinases was monitored at 345 nm in the presence of increasing GdnHCl concentration (Figure 3). Using this technique, a similar pattern of stability was observed to that of thermal unfolding. EphB1 appeared to tolerate a higher concentration of GdnHCl than the other enzymes before beginning to unfold. EphB2 appeared to start unfolding at a lower GdnHCl concentration, while EphB3 and EphB4 were both markedly less tolerant to chaotrope concentration. At a GdnHCl concentration of 3 M, a stable fluorescence minimum for all four kinase domains was reached, indicating complete unfolding. Each of the four proteins appears to fit more closely to a three-state unfolding model than a two-state model (Table 2), with evidence of a partially stable intermediate unfolding state at about 2 M GdnHCl, which appears more pronounced in EphB3 and EphB4, indicating a stable core common to each of the kinases. The most striking observation between the unfolding curves of the four proteins is the apparently ‘unfolded’ state observed at ~1 M GuHCl with EphB3: at this point, the tryptophan emission signal almost disappears and then reappears again at 1.5 M GuHCl.


Biochemical and biophysical characterization of four EphB kinase domains reveals contrasting thermodynamic, kinetic and inhibition profiles.

Overman RC, Debreczeni JE, Truman CM, McAlister MS, Attwood TK - Biosci. Rep. (2013)

Chaotrope-induced denaturation of EphB kinaseUnfolding EphB kinases in the presence of increasing GdnHCl was monitored by following the progressive quenching of internal tryptophan fluorescence (excitation 295 nm, emission 345 nm), as described. The y-axis shows relative fluorescence units measured at 345 nm averaged from five scans of three independent experiments. Curves were fitted to both two-state (broken line) and three-state (unbroken line) models [28,29], using Prism software (GraphPad Software, Inc.).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3673036&req=5

Figure 3: Chaotrope-induced denaturation of EphB kinaseUnfolding EphB kinases in the presence of increasing GdnHCl was monitored by following the progressive quenching of internal tryptophan fluorescence (excitation 295 nm, emission 345 nm), as described. The y-axis shows relative fluorescence units measured at 345 nm averaged from five scans of three independent experiments. Curves were fitted to both two-state (broken line) and three-state (unbroken line) models [28,29], using Prism software (GraphPad Software, Inc.).
Mentions: In an attempt to follow the unfolding events of each of the four kinases in greater detail, chaotrope-induced unfolding was performed. Internal tryptophan fluorescence of the four kinases was monitored at 345 nm in the presence of increasing GdnHCl concentration (Figure 3). Using this technique, a similar pattern of stability was observed to that of thermal unfolding. EphB1 appeared to tolerate a higher concentration of GdnHCl than the other enzymes before beginning to unfold. EphB2 appeared to start unfolding at a lower GdnHCl concentration, while EphB3 and EphB4 were both markedly less tolerant to chaotrope concentration. At a GdnHCl concentration of 3 M, a stable fluorescence minimum for all four kinase domains was reached, indicating complete unfolding. Each of the four proteins appears to fit more closely to a three-state unfolding model than a two-state model (Table 2), with evidence of a partially stable intermediate unfolding state at about 2 M GdnHCl, which appears more pronounced in EphB3 and EphB4, indicating a stable core common to each of the kinases. The most striking observation between the unfolding curves of the four proteins is the apparently ‘unfolded’ state observed at ~1 M GuHCl with EphB3: at this point, the tryptophan emission signal almost disappears and then reappears again at 1.5 M GuHCl.

Bottom Line: A greater understanding of the similarities and differences within this small, highly conserved family of tyrosine kinases will be essential to the identification of effective therapeutic opportunities for disease intervention.Our findings have led us to speculate about both their biological significance and potential routes for generating EphB isozyme-selective small-molecule inhibitors.Our comprehensive methodologies provide a template for similar in-depth studies of other kinase superfamily members.

View Article: PubMed Central - PubMed

Affiliation: AstraZeneca PLC, Alderley Park, Cheshire, SK10 4TG, UK. ross.overman@astrazeneca.com

ABSTRACT
The Eph (erythropoietin-producing hepatocellular carcinoma) B receptors are important in a variety of cellular processes through their roles in cell-to-cell contact and signalling; their up-regulation and down-regulation has been shown to have implications in a variety of cancers. A greater understanding of the similarities and differences within this small, highly conserved family of tyrosine kinases will be essential to the identification of effective therapeutic opportunities for disease intervention. In this study, we have developed a route to production of multi-milligram quantities of highly purified, homogeneous, recombinant protein for the kinase domain of these human receptors in Escherichia coli. Analyses of these isolated catalytic fragments have revealed stark contrasts in their amenability to recombinant expression and their physical properties: e.g., a >16°C variance in thermal stability, a 3-fold difference in catalytic activity and disparities in their inhibitor binding profiles. We find EphB3 to be an outlier in terms of both its intrinsic stability, and more importantly its ligand-binding properties. Our findings have led us to speculate about both their biological significance and potential routes for generating EphB isozyme-selective small-molecule inhibitors. Our comprehensive methodologies provide a template for similar in-depth studies of other kinase superfamily members.

Show MeSH
Related in: MedlinePlus