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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

Thermal stability analyses using CD spectroscopyRepresentative unfolding transitions obtained for each of the four kinases from thermal denaturation experiments monitored using CD spectroscopy at 222 nm (α-helical response). Unfolding was conducted using 10 μM kinase at pH 7.4 under reducing conditions.
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Figure 2: Thermal stability analyses using CD spectroscopyRepresentative unfolding transitions obtained for each of the four kinases from thermal denaturation experiments monitored using CD spectroscopy at 222 nm (α-helical response). Unfolding was conducted using 10 μM kinase at pH 7.4 under reducing conditions.

Mentions: To investigate whether observed differences in soluble, recombinant expression levels in E. coli could be attributed to differences in stability between the isolated kinase domains, thermal unfolding events for each of the four kinase domains were studied using CD spectroscopy. Far UV wavelength scans were performed in phosphate buffer at physiological pH, and demonstrated very similar secondary structure profiles, as would be expected given the level of sequence and structural identity between the four domains (Supplementary Figure S2 available at http://www.bioscirep.org/bsr/033/bsr033e040add.htm). Strong α-helical signatures for all four proteins allowed temperature-dependent unfolding to be monitored at 222 nm. A two-state unfolding transition was observed for each protein over a 20–80°C range (Figure 2), enabling melting temperatures (Tm, midpoint of unfolding) to be determined for all four kinases in their unphosphorylated forms (Table 2). A striking difference between the melting temperatures of the EphB kinase domains was observed, which coincidentally rank in order of their numbering, with EphB1 being the most stable, and a difference between apparent melting temperature for EphB1 and EphB4 of 16.9°C. The differences in thermal stability observed by CD unfolding were further confirmed using DSF (differential scanning fluorimetry) [33] (Supplementary Figure S3 available at http://www.bioscirep.org/bsr/033/bsr033e040add.htm).


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)

Thermal stability analyses using CD spectroscopyRepresentative unfolding transitions obtained for each of the four kinases from thermal denaturation experiments monitored using CD spectroscopy at 222 nm (α-helical response). Unfolding was conducted using 10 μM kinase at pH 7.4 under reducing conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Thermal stability analyses using CD spectroscopyRepresentative unfolding transitions obtained for each of the four kinases from thermal denaturation experiments monitored using CD spectroscopy at 222 nm (α-helical response). Unfolding was conducted using 10 μM kinase at pH 7.4 under reducing conditions.
Mentions: To investigate whether observed differences in soluble, recombinant expression levels in E. coli could be attributed to differences in stability between the isolated kinase domains, thermal unfolding events for each of the four kinase domains were studied using CD spectroscopy. Far UV wavelength scans were performed in phosphate buffer at physiological pH, and demonstrated very similar secondary structure profiles, as would be expected given the level of sequence and structural identity between the four domains (Supplementary Figure S2 available at http://www.bioscirep.org/bsr/033/bsr033e040add.htm). Strong α-helical signatures for all four proteins allowed temperature-dependent unfolding to be monitored at 222 nm. A two-state unfolding transition was observed for each protein over a 20–80°C range (Figure 2), enabling melting temperatures (Tm, midpoint of unfolding) to be determined for all four kinases in their unphosphorylated forms (Table 2). A striking difference between the melting temperatures of the EphB kinase domains was observed, which coincidentally rank in order of their numbering, with EphB1 being the most stable, and a difference between apparent melting temperature for EphB1 and EphB4 of 16.9°C. The differences in thermal stability observed by CD unfolding were further confirmed using DSF (differential scanning fluorimetry) [33] (Supplementary Figure S3 available at http://www.bioscirep.org/bsr/033/bsr033e040add.htm).

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