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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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G7-18NATE binding surface on the Grb7 SH2 domain. (a) An overlay of the 15N, 1H-HSQC spectra of Grb7 SH2 alone (black) and in the presence of two molar equivalents of G7-18NATE (red). The insert shows changes to signals in the boxed region of the HSQC spectrum over the course of the titration. The ratio of G7-18NATE to Grb7 SH2 is 0:1, 1:4, 1:2, 3:4, 1:1 and 2:1 in the black, red, blue, orange, purple and green spectra respectively. (b) The rate of change of peak volume over the titration series versus Grb7 SH2 domain residue number. The broken line indicates the mean rate of change. Residues that exhibited rates of change greater than this value are coloured according to their location in the Grb7 SH2 domain structure. (c) Surface representation of the Grb7 SH2 domain with residues affected by G7-18NATE binding coloured. The majority of residues cluster onto either the phosphopeptide binding surface (red) or the dimerisation interface (green). Residues shown in blue were also affected by G7-18NATE binding but do not cluster on one surface. The two images are related by a 90° rotation about the horizontal axis of the page.
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Figure 6: G7-18NATE binding surface on the Grb7 SH2 domain. (a) An overlay of the 15N, 1H-HSQC spectra of Grb7 SH2 alone (black) and in the presence of two molar equivalents of G7-18NATE (red). The insert shows changes to signals in the boxed region of the HSQC spectrum over the course of the titration. The ratio of G7-18NATE to Grb7 SH2 is 0:1, 1:4, 1:2, 3:4, 1:1 and 2:1 in the black, red, blue, orange, purple and green spectra respectively. (b) The rate of change of peak volume over the titration series versus Grb7 SH2 domain residue number. The broken line indicates the mean rate of change. Residues that exhibited rates of change greater than this value are coloured according to their location in the Grb7 SH2 domain structure. (c) Surface representation of the Grb7 SH2 domain with residues affected by G7-18NATE binding coloured. The majority of residues cluster onto either the phosphopeptide binding surface (red) or the dimerisation interface (green). Residues shown in blue were also affected by G7-18NATE binding but do not cluster on one surface. The two images are related by a 90° rotation about the horizontal axis of the page.

Mentions: In order to further characterize the Grb7 SH2 domain/G7-18NATE interaction NMR titration experiments were used to identify sites of Grb7 SH2 perturbation upon binding by the G7-18NATE peptide. 1H-15N-HSQC spectra were acquired for 15N-labelled Grb7 SH2 as G7-18NATE was titrated in to slight excess (Figure 6A). Approximately 80% of the amide crosspeaks exhibited line broadening, consistent with an 'intermediate' rate of exchange of free and bound forms of the protein (where the exchange rate is close to the chemical shift differences of the resonances between free and bound forms). Other peaks remained unperturbed throughout the titration or exhibited a chemical shift change rather than linebroadening. This behavior is common for interactions for which the dissociation constant is between 10-3 and 10-6 M.


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)

G7-18NATE binding surface on the Grb7 SH2 domain. (a) An overlay of the 15N, 1H-HSQC spectra of Grb7 SH2 alone (black) and in the presence of two molar equivalents of G7-18NATE (red). The insert shows changes to signals in the boxed region of the HSQC spectrum over the course of the titration. The ratio of G7-18NATE to Grb7 SH2 is 0:1, 1:4, 1:2, 3:4, 1:1 and 2:1 in the black, red, blue, orange, purple and green spectra respectively. (b) The rate of change of peak volume over the titration series versus Grb7 SH2 domain residue number. The broken line indicates the mean rate of change. Residues that exhibited rates of change greater than this value are coloured according to their location in the Grb7 SH2 domain structure. (c) Surface representation of the Grb7 SH2 domain with residues affected by G7-18NATE binding coloured. The majority of residues cluster onto either the phosphopeptide binding surface (red) or the dimerisation interface (green). Residues shown in blue were also affected by G7-18NATE binding but do not cluster on one surface. The two images are related by a 90° rotation about the horizontal axis of the page.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: G7-18NATE binding surface on the Grb7 SH2 domain. (a) An overlay of the 15N, 1H-HSQC spectra of Grb7 SH2 alone (black) and in the presence of two molar equivalents of G7-18NATE (red). The insert shows changes to signals in the boxed region of the HSQC spectrum over the course of the titration. The ratio of G7-18NATE to Grb7 SH2 is 0:1, 1:4, 1:2, 3:4, 1:1 and 2:1 in the black, red, blue, orange, purple and green spectra respectively. (b) The rate of change of peak volume over the titration series versus Grb7 SH2 domain residue number. The broken line indicates the mean rate of change. Residues that exhibited rates of change greater than this value are coloured according to their location in the Grb7 SH2 domain structure. (c) Surface representation of the Grb7 SH2 domain with residues affected by G7-18NATE binding coloured. The majority of residues cluster onto either the phosphopeptide binding surface (red) or the dimerisation interface (green). Residues shown in blue were also affected by G7-18NATE binding but do not cluster on one surface. The two images are related by a 90° rotation about the horizontal axis of the page.
Mentions: In order to further characterize the Grb7 SH2 domain/G7-18NATE interaction NMR titration experiments were used to identify sites of Grb7 SH2 perturbation upon binding by the G7-18NATE peptide. 1H-15N-HSQC spectra were acquired for 15N-labelled Grb7 SH2 as G7-18NATE was titrated in to slight excess (Figure 6A). Approximately 80% of the amide crosspeaks exhibited line broadening, consistent with an 'intermediate' rate of exchange of free and bound forms of the protein (where the exchange rate is close to the chemical shift differences of the resonances between free and bound forms). Other peaks remained unperturbed throughout the titration or exhibited a chemical shift change rather than linebroadening. This behavior is common for interactions for which the dissociation constant is between 10-3 and 10-6 M.

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