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Published on: October 30, 2012
Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation
Corrine J Porter1, Jacqueline M Matthews, Joel P Mackay
1School of Biomedical and Chemical Sciences, University of Western Australia, WA 6009, Australia. Corrine.Porter@med.monash.edu.au
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.
Insights
An inhibitory peptide (G7-18NATE) targets the Grb7 SH2 domain, crucial for cancer progression. Structural and binding studies reveal how this peptide inhibits Grb7, paving the way for new anti-cancer therapies targeting cell migration.
Area of Science:
- Biochemistry
- Structural Biology
- Cancer Research
Background:
- Human growth factor receptor bound protein 7 (Grb7) is an adapter protein overexpressed in invasive and metastatic cancers.
- Grb7 plays a critical role in cancer progression by interacting with ErbB2 and FAK, influencing cell proliferation and migration.
- An inhibitory peptide, G7-18NATE, specifically targets the Grb7 SH2 domain to attenuate cancer cell functions.
Purpose of the Study:
- To elucidate the structural basis of G7-18NATE inhibition of the Grb7 SH2 domain.
- To understand the molecular interactions governing Grb7 SH2 domain binding specificity and affinity.
- To provide a foundation for developing second-generation Grb7 inhibitors for cancer therapy.
Main Methods:
- Crystal structure determination of the Grb7 SH2 domain at 2.1 A resolution.
- Analytical ultracentrifugation to assess Grb7 SH2 domain dimerization in the microM range.
- Isothermal Titration Calorimetry (ITC) and NMR spectroscopy to characterize G7-18NATE peptide binding to the Grb7 SH2 domain.
Main Results:
- The crystal structure reveals the peptide binding site and the Grb7 SH2 dimer interface.
- Grb7 SH2 domain forms a physiological dimer, with dissociation constants in the microM range.
- G7-18NATE binds the Grb7 SH2 domain with a Kd of approximately 35.7 microM, perturbing both the ligand-binding surface and the dimer interface.
Conclusions:
- The study proposes a model for the Grb7 SH2 domain/G7-18NATE interaction, explaining binding specificity and affinity.
- Peptide binding impacts Grb7 SH2 domain dimerization, offering a mechanism for inhibition.
- These findings will aid in developing novel Grb7 SH2 domain inhibitors to combat cancer cell migration and invasion.
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