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Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation.

Porter CJ, Matthews JM, Mackay JP, Pursglove SE, Schmidberger JW, Leedman PJ, Pero SC, Krag DN, Wilce MC, Wilce JA - BMC Struct. Biol. (2007)

Bottom Line: Dimer formation of Grb7 was determined to be in the muM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer.ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of Kd = approximately 35.7 microM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biomedical and Chemical Sciences, University of Western Australia, WA 6009, Australia. Corrine.Porter@med.monash.edu.au

ABSTRACT

Background: Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

Results: As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 A resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the muM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of Kd = approximately 35.7 microM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.

Conclusion: Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.

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Isothermal titration calorimetric measurement of G7-18NATE peptide binding to the Grb7 SH2 domain (a) The sequence of the G7-18NATE peptide used in this study. (b) ITC data showing the titration of G7-18NATE into Grb7 SH2. The samples were prepared in 50 mM MES pH 6.6, 100 mM NaCl and 1 mM DTT. ITC experiments were carried out in duplicate at 25°C with Grb7 SH2 in the cell at 25 μM and G7-18NATE in the syringe at 440 μM. The upper panel shows the raw data while the lower panel shows the data after peak integration and subtraction of the heat-of-dilution control. In this panel the grey line represents the fit to a 1:1 binding model.
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Figure 5: Isothermal titration calorimetric measurement of G7-18NATE peptide binding to the Grb7 SH2 domain (a) The sequence of the G7-18NATE peptide used in this study. (b) ITC data showing the titration of G7-18NATE into Grb7 SH2. The samples were prepared in 50 mM MES pH 6.6, 100 mM NaCl and 1 mM DTT. ITC experiments were carried out in duplicate at 25°C with Grb7 SH2 in the cell at 25 μM and G7-18NATE in the syringe at 440 μM. The upper panel shows the raw data while the lower panel shows the data after peak integration and subtraction of the heat-of-dilution control. In this panel the grey line represents the fit to a 1:1 binding model.

Mentions: The G7-18NATE peptide (Figure 5A) is the first non-phosphorylated peptide to be discovered with binding specificity for the Grb7 SH2 domain. It has been found to bind to the Grb7 SH2 domain, preferentially over the Grb14 SH2 domain, and have the capacity to interfere with Grb7 interactions with the ErbB family of receptors [28] and FAK in vivo [20]. These interactions may underlie the observed inhibition of breast cancer cell proliferation and decrease in cell migration of pancreatic cancer cells treated with G7-18NATE, making it of considerable importance to further characterise this peptide and its interactions with Grb7 SH2 domain. Isothermal titration calorimetry was thus used to determine the binding affinity of G7-18NATE for the Grb7 SH2 domain (Figure 5B). This data showed that the peptide bound to the SH2 domain with an affinity of Kd = ~35.7 μ M. This is only an order of magnitude less than the Kd = 2.3 μM measured by Lyons and co-workers for the binding of the Grb7 SH2 domain to pY1139, a native phosphopeptide ligand derived from the ErbB2 receptor [39].


Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation.

Porter CJ, Matthews JM, Mackay JP, Pursglove SE, Schmidberger JW, Leedman PJ, Pero SC, Krag DN, Wilce MC, Wilce JA - BMC Struct. Biol. (2007)

Isothermal titration calorimetric measurement of G7-18NATE peptide binding to the Grb7 SH2 domain (a) The sequence of the G7-18NATE peptide used in this study. (b) ITC data showing the titration of G7-18NATE into Grb7 SH2. The samples were prepared in 50 mM MES pH 6.6, 100 mM NaCl and 1 mM DTT. ITC experiments were carried out in duplicate at 25°C with Grb7 SH2 in the cell at 25 μM and G7-18NATE in the syringe at 440 μM. The upper panel shows the raw data while the lower panel shows the data after peak integration and subtraction of the heat-of-dilution control. In this panel the grey line represents the fit to a 1:1 binding model.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2131756&req=5

Figure 5: Isothermal titration calorimetric measurement of G7-18NATE peptide binding to the Grb7 SH2 domain (a) The sequence of the G7-18NATE peptide used in this study. (b) ITC data showing the titration of G7-18NATE into Grb7 SH2. The samples were prepared in 50 mM MES pH 6.6, 100 mM NaCl and 1 mM DTT. ITC experiments were carried out in duplicate at 25°C with Grb7 SH2 in the cell at 25 μM and G7-18NATE in the syringe at 440 μM. The upper panel shows the raw data while the lower panel shows the data after peak integration and subtraction of the heat-of-dilution control. In this panel the grey line represents the fit to a 1:1 binding model.
Mentions: The G7-18NATE peptide (Figure 5A) is the first non-phosphorylated peptide to be discovered with binding specificity for the Grb7 SH2 domain. It has been found to bind to the Grb7 SH2 domain, preferentially over the Grb14 SH2 domain, and have the capacity to interfere with Grb7 interactions with the ErbB family of receptors [28] and FAK in vivo [20]. These interactions may underlie the observed inhibition of breast cancer cell proliferation and decrease in cell migration of pancreatic cancer cells treated with G7-18NATE, making it of considerable importance to further characterise this peptide and its interactions with Grb7 SH2 domain. Isothermal titration calorimetry was thus used to determine the binding affinity of G7-18NATE for the Grb7 SH2 domain (Figure 5B). This data showed that the peptide bound to the SH2 domain with an affinity of Kd = ~35.7 μ M. This is only an order of magnitude less than the Kd = 2.3 μM measured by Lyons and co-workers for the binding of the Grb7 SH2 domain to pY1139, a native phosphopeptide ligand derived from the ErbB2 receptor [39].

Bottom Line: Dimer formation of Grb7 was determined to be in the muM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer.ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of Kd = approximately 35.7 microM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biomedical and Chemical Sciences, University of Western Australia, WA 6009, Australia. Corrine.Porter@med.monash.edu.au

ABSTRACT

Background: Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

Results: As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 A resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the muM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of Kd = approximately 35.7 microM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.

Conclusion: Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.

Show MeSH
Related in: MedlinePlus