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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: We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2.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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Sedimentation equilibrium analysis of the Grb7 SH2 and Grb7. (a) Absorbance at 280 nm verses radius data at sedimentation equilibrium for Grb7 SH2 at an initial loading concentration of 36 μM. The data collected at 14,000 rpm (circles), 16,600 rpm (squares), 24,300 rpm (diamonds) and 28,800 rpm (triangles) were fitted simultaneously using the nonlinear regression program [48]. (b) Absorbance at 280 nm verses radius data at sedimentation equilibrium for Grb7 at an initial loading concentrations of 12 μM. The data collected at 10,000 rpm (circles) and 11,800 rpm (squares) were fitted simultaneously using the nonlinear regression program NONLIN [77]. The solid line represents the calculated fit to a monomer-dimer model. The residuals of the fit are shown in the upper panels. Samples were in 50 mM MES pH 6.6, 100 mM NaCl and 1 mM DTT. The experiments were conducted at 20°C.
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Figure 4: Sedimentation equilibrium analysis of the Grb7 SH2 and Grb7. (a) Absorbance at 280 nm verses radius data at sedimentation equilibrium for Grb7 SH2 at an initial loading concentration of 36 μM. The data collected at 14,000 rpm (circles), 16,600 rpm (squares), 24,300 rpm (diamonds) and 28,800 rpm (triangles) were fitted simultaneously using the nonlinear regression program [48]. (b) Absorbance at 280 nm verses radius data at sedimentation equilibrium for Grb7 at an initial loading concentrations of 12 μM. The data collected at 10,000 rpm (circles) and 11,800 rpm (squares) were fitted simultaneously using the nonlinear regression program NONLIN [77]. The solid line represents the calculated fit to a monomer-dimer model. The residuals of the fit are shown in the upper panels. Samples were in 50 mM MES pH 6.6, 100 mM NaCl and 1 mM DTT. The experiments were conducted at 20°C.

Mentions: The observation of dimer formation in the crystal structure prompted us to investigate the strength of the interaction between Grb7 SH2 domains as well as between full-length Grb7 molecules. The dimer dissociation constant was calculated for both the Grb7 SH2 domain and full-length Grb7 at 20°C using analytical ultracentrifugation. Figure 4 shows profiles recorded for the two proteins that could be fitted globally to a monomer-dimer self-association model using NONLIN [48,49]. This yielded a dissociation equilibrium constant for the Grb7 SH2 domain of 21.8 μM. This represents a slightly weaker association than that determined in previously reported experiments conducted at 4°C [38], consistent with the lesser role of hydrophobic interactions at the higher temperature. The profiles obtained for full-length Grb7 at 12 μM yielded a dissociation equilibrium constant of 11 μM. Although some sample heterogeneity, possibly due to protein aggregation or degradation, prevented a simultaneous fit with profiles recorded at higher concentrations, the data demonstrated that full-length Grb7 forms dimers in vitro with a similar affinity to that exhibited by the SH2 domain alone. This suggests that the Grb7 molecule may exist as a dimer in vivo via its SH2 domain, as also thought to occur for the Grb10 and Grb14 molecules [36,37].


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)

Sedimentation equilibrium analysis of the Grb7 SH2 and Grb7. (a) Absorbance at 280 nm verses radius data at sedimentation equilibrium for Grb7 SH2 at an initial loading concentration of 36 μM. The data collected at 14,000 rpm (circles), 16,600 rpm (squares), 24,300 rpm (diamonds) and 28,800 rpm (triangles) were fitted simultaneously using the nonlinear regression program [48]. (b) Absorbance at 280 nm verses radius data at sedimentation equilibrium for Grb7 at an initial loading concentrations of 12 μM. The data collected at 10,000 rpm (circles) and 11,800 rpm (squares) were fitted simultaneously using the nonlinear regression program NONLIN [77]. The solid line represents the calculated fit to a monomer-dimer model. The residuals of the fit are shown in the upper panels. Samples were in 50 mM MES pH 6.6, 100 mM NaCl and 1 mM DTT. The experiments were conducted at 20°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Sedimentation equilibrium analysis of the Grb7 SH2 and Grb7. (a) Absorbance at 280 nm verses radius data at sedimentation equilibrium for Grb7 SH2 at an initial loading concentration of 36 μM. The data collected at 14,000 rpm (circles), 16,600 rpm (squares), 24,300 rpm (diamonds) and 28,800 rpm (triangles) were fitted simultaneously using the nonlinear regression program [48]. (b) Absorbance at 280 nm verses radius data at sedimentation equilibrium for Grb7 at an initial loading concentrations of 12 μM. The data collected at 10,000 rpm (circles) and 11,800 rpm (squares) were fitted simultaneously using the nonlinear regression program NONLIN [77]. The solid line represents the calculated fit to a monomer-dimer model. The residuals of the fit are shown in the upper panels. Samples were in 50 mM MES pH 6.6, 100 mM NaCl and 1 mM DTT. The experiments were conducted at 20°C.
Mentions: The observation of dimer formation in the crystal structure prompted us to investigate the strength of the interaction between Grb7 SH2 domains as well as between full-length Grb7 molecules. The dimer dissociation constant was calculated for both the Grb7 SH2 domain and full-length Grb7 at 20°C using analytical ultracentrifugation. Figure 4 shows profiles recorded for the two proteins that could be fitted globally to a monomer-dimer self-association model using NONLIN [48,49]. This yielded a dissociation equilibrium constant for the Grb7 SH2 domain of 21.8 μM. This represents a slightly weaker association than that determined in previously reported experiments conducted at 4°C [38], consistent with the lesser role of hydrophobic interactions at the higher temperature. The profiles obtained for full-length Grb7 at 12 μM yielded a dissociation equilibrium constant of 11 μM. Although some sample heterogeneity, possibly due to protein aggregation or degradation, prevented a simultaneous fit with profiles recorded at higher concentrations, the data demonstrated that full-length Grb7 forms dimers in vitro with a similar affinity to that exhibited by the SH2 domain alone. This suggests that the Grb7 molecule may exist as a dimer in vivo via its SH2 domain, as also thought to occur for the Grb10 and Grb14 molecules [36,37].

Bottom Line: We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2.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