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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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The phosphate binding pocket of the Grb7 SH2 domain. (a) Electrostatic potential surface of Grb7 SH2 generated using CCP4MG [80]. Positively charged electrostatic potential is coloured blue and negatively charged electrostatic potential is coloured red. The positions of the phosphate binding pocket is indicated. (b) A 2Fo - Fc electron density map depicting the phosphate binding pocket of Grb7 SH2. A sulphate ion co-crystallised in this pocket in all four molecules in the asymmetric unit. The map is contoured at 1 σ. R438, R458, Q461 and S460 form direct contacts with the sulphate ion and are labeled. The side-chain of R462 lacks well defined density and is probably fairly mobile in the crystal.
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Figure 2: The phosphate binding pocket of the Grb7 SH2 domain. (a) Electrostatic potential surface of Grb7 SH2 generated using CCP4MG [80]. Positively charged electrostatic potential is coloured blue and negatively charged electrostatic potential is coloured red. The positions of the phosphate binding pocket is indicated. (b) A 2Fo - Fc electron density map depicting the phosphate binding pocket of Grb7 SH2. A sulphate ion co-crystallised in this pocket in all four molecules in the asymmetric unit. The map is contoured at 1 σ. R438, R458, Q461 and S460 form direct contacts with the sulphate ion and are labeled. The side-chain of R462 lacks well defined density and is probably fairly mobile in the crystal.

Mentions: The ligand binding site within SH2 domains comprises the phosphotyrosine (pY) binding pocket and adjacent peptide binding site that confers specificity for the residues immediately C-terminal to the pY [40]. The Grb7 SH2 pY binding pocket exists in a cleft formed by residues in the central β-sheet, the αA and the BC loop (also referred to as the phosphate binding loop) and possesses a large positive potential for interacting with the phosphotyrosine (Figure 2A). Although the BC loop within apo-SH2 domain structures usually exhibits poorly defined electron density [25], the Grb7 SH2 domain BC loop is well defined and closely resembles that of SH2 domains in their peptide bound conformation. The BC loop is held in this conformation by a complex network of hydrogen bond contacts formed with a sulphate anion that appears to mimic the phosphate moiety of the natural ligand (Figure 2B). These contacts include a bidentate ionic interaction with the side-chain of the invariant Arg 458 (βB5). Additional hydrogen bonds are formed with the side-chains of Arg 438 (αA2), Ser 460 (βB7) and the main-chain amide of Gln 461 (BC1).


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)

The phosphate binding pocket of the Grb7 SH2 domain. (a) Electrostatic potential surface of Grb7 SH2 generated using CCP4MG [80]. Positively charged electrostatic potential is coloured blue and negatively charged electrostatic potential is coloured red. The positions of the phosphate binding pocket is indicated. (b) A 2Fo - Fc electron density map depicting the phosphate binding pocket of Grb7 SH2. A sulphate ion co-crystallised in this pocket in all four molecules in the asymmetric unit. The map is contoured at 1 σ. R438, R458, Q461 and S460 form direct contacts with the sulphate ion and are labeled. The side-chain of R462 lacks well defined density and is probably fairly mobile in the crystal.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The phosphate binding pocket of the Grb7 SH2 domain. (a) Electrostatic potential surface of Grb7 SH2 generated using CCP4MG [80]. Positively charged electrostatic potential is coloured blue and negatively charged electrostatic potential is coloured red. The positions of the phosphate binding pocket is indicated. (b) A 2Fo - Fc electron density map depicting the phosphate binding pocket of Grb7 SH2. A sulphate ion co-crystallised in this pocket in all four molecules in the asymmetric unit. The map is contoured at 1 σ. R438, R458, Q461 and S460 form direct contacts with the sulphate ion and are labeled. The side-chain of R462 lacks well defined density and is probably fairly mobile in the crystal.
Mentions: The ligand binding site within SH2 domains comprises the phosphotyrosine (pY) binding pocket and adjacent peptide binding site that confers specificity for the residues immediately C-terminal to the pY [40]. The Grb7 SH2 pY binding pocket exists in a cleft formed by residues in the central β-sheet, the αA and the BC loop (also referred to as the phosphate binding loop) and possesses a large positive potential for interacting with the phosphotyrosine (Figure 2A). Although the BC loop within apo-SH2 domain structures usually exhibits poorly defined electron density [25], the Grb7 SH2 domain BC loop is well defined and closely resembles that of SH2 domains in their peptide bound conformation. The BC loop is held in this conformation by a complex network of hydrogen bond contacts formed with a sulphate anion that appears to mimic the phosphate moiety of the natural ligand (Figure 2B). These contacts include a bidentate ionic interaction with the side-chain of the invariant Arg 458 (βB5). Additional hydrogen bonds are formed with the side-chains of Arg 438 (αA2), Ser 460 (βB7) and the main-chain amide of Gln 461 (BC1).

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