Limits...
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.

Show MeSH

Related in: MedlinePlus

Model of the Grb7 SH2/G7-18NATE complex. (a) The modeled structure of the complex. The Grb7 SH2 domain is shown in grey in cartoon representation and G7-18NATE in stick representation with carbon atoms coloured green. The positions of the two α-helices are indicated. The model was prepared using the structure of the Grb2 SH2:pYVN complex (1JYR; [52]) as a template and energy minimised using NAMD [79]. (b) A close up of the Grb7 SH2/G718-NATE interface. The Glu3 and Tyr5 side-chains occupy the phosphate binding pocket while the Asn7 side-chain occupies a second pocket in the specificity determining region of the domain. Only Glu3, Tyr5, Asp6 and Asn7 are shown for clarity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2131756&req=5

Figure 7: Model of the Grb7 SH2/G7-18NATE complex. (a) The modeled structure of the complex. The Grb7 SH2 domain is shown in grey in cartoon representation and G7-18NATE in stick representation with carbon atoms coloured green. The positions of the two α-helices are indicated. The model was prepared using the structure of the Grb2 SH2:pYVN complex (1JYR; [52]) as a template and energy minimised using NAMD [79]. (b) A close up of the Grb7 SH2/G718-NATE interface. The Glu3 and Tyr5 side-chains occupy the phosphate binding pocket while the Asn7 side-chain occupies a second pocket in the specificity determining region of the domain. Only Glu3, Tyr5, Asp6 and Asn7 are shown for clarity.

Mentions: Together the structural data has allowed a model of the G7-18NATE peptide interacting with the Grb7 SH2 domain to be constructed. In separate studies we have shown that the G7-18NATE peptide has an intrinsic propensity for a turn conformation about the YDN motif [51]. The current study also confirmed that G7-18NATE binding impacts on the Grb7 SH2 domain surface in a similar way to the pY1139 peptide, and is thus likely to bind at the classical peptide binding site. A model of the G7-18NATE peptide was hence positioned at the peptide binding site of the crystallographically determined Grb7 SH2 domain structure by analogy to the Shc derived peptide (PSpYVNVQN) which interacts with Grb 2 SH2 domain in a turn conformation (1JYR [52]) (Figure 7). In this model Asn7, in the +2 position relative to Tyr5, is positioned to form hydrogen bond interactions via its sidechain carbonyl and amine functionalities with backbone NH and carbonyl groups of the βD6 residue. The non-phosphorylated tyrosine (Tyr5) is able to adopt a position in which its hydroxyl is positioned in the positively charged pY binding site. The aromatic ring of Tyr 5 forms Van der Waals contacts with the βD6 residue sidechain (Leu 481) that may be important for the positioning of the peptide ligand. Glu3 is also positioned in the pY binding site. Electrostatic interactions formed by the Glu3 sidechain may compensate for the lack of a phosphate moiety on the tyrosine. The side chain of Asp6 is oriented away from the binding cleft and is in proximity of the backbone NH of the βD8 (His483) residue with which it could form a hydrogen bond. Other residues, constrained in a cyclical conformation, may make contacts at the surface of βC, βD' and EF loop residues shown to be perturbed by NMR spectroscopy. Whilst this model should remain speculative, it suggests a likely basis for the relatively high affinity and specific interaction between G7-18NATE and the Grb7 SH2 domain.


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)

Model of the Grb7 SH2/G7-18NATE complex. (a) The modeled structure of the complex. The Grb7 SH2 domain is shown in grey in cartoon representation and G7-18NATE in stick representation with carbon atoms coloured green. The positions of the two α-helices are indicated. The model was prepared using the structure of the Grb2 SH2:pYVN complex (1JYR; [52]) as a template and energy minimised using NAMD [79]. (b) A close up of the Grb7 SH2/G718-NATE interface. The Glu3 and Tyr5 side-chains occupy the phosphate binding pocket while the Asn7 side-chain occupies a second pocket in the specificity determining region of the domain. Only Glu3, Tyr5, Asp6 and Asn7 are shown for clarity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Model of the Grb7 SH2/G7-18NATE complex. (a) The modeled structure of the complex. The Grb7 SH2 domain is shown in grey in cartoon representation and G7-18NATE in stick representation with carbon atoms coloured green. The positions of the two α-helices are indicated. The model was prepared using the structure of the Grb2 SH2:pYVN complex (1JYR; [52]) as a template and energy minimised using NAMD [79]. (b) A close up of the Grb7 SH2/G718-NATE interface. The Glu3 and Tyr5 side-chains occupy the phosphate binding pocket while the Asn7 side-chain occupies a second pocket in the specificity determining region of the domain. Only Glu3, Tyr5, Asp6 and Asn7 are shown for clarity.
Mentions: Together the structural data has allowed a model of the G7-18NATE peptide interacting with the Grb7 SH2 domain to be constructed. In separate studies we have shown that the G7-18NATE peptide has an intrinsic propensity for a turn conformation about the YDN motif [51]. The current study also confirmed that G7-18NATE binding impacts on the Grb7 SH2 domain surface in a similar way to the pY1139 peptide, and is thus likely to bind at the classical peptide binding site. A model of the G7-18NATE peptide was hence positioned at the peptide binding site of the crystallographically determined Grb7 SH2 domain structure by analogy to the Shc derived peptide (PSpYVNVQN) which interacts with Grb 2 SH2 domain in a turn conformation (1JYR [52]) (Figure 7). In this model Asn7, in the +2 position relative to Tyr5, is positioned to form hydrogen bond interactions via its sidechain carbonyl and amine functionalities with backbone NH and carbonyl groups of the βD6 residue. The non-phosphorylated tyrosine (Tyr5) is able to adopt a position in which its hydroxyl is positioned in the positively charged pY binding site. The aromatic ring of Tyr 5 forms Van der Waals contacts with the βD6 residue sidechain (Leu 481) that may be important for the positioning of the peptide ligand. Glu3 is also positioned in the pY binding site. Electrostatic interactions formed by the Glu3 sidechain may compensate for the lack of a phosphate moiety on the tyrosine. The side chain of Asp6 is oriented away from the binding cleft and is in proximity of the backbone NH of the βD8 (His483) residue with which it could form a hydrogen bond. Other residues, constrained in a cyclical conformation, may make contacts at the surface of βC, βD' and EF loop residues shown to be perturbed by NMR spectroscopy. Whilst this model should remain speculative, it suggests a likely basis for the relatively high affinity and specific interaction between G7-18NATE and the Grb7 SH2 domain.

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