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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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Crystal structure of the Grb7 SH2 domain. (a) Structure based sequence alignment of Grb7 SH2, Grb14 SH2 (2AUG; [37]) and Grb10 SH2 (1NRV; [36]). Secondary structure elements present in the Grb7 SH2 structure as determined by WHATIF [71] are shaded from purple at the N-terminus to red at the C-terminus. Secondary structure elements of the canonical SH2 domain as defined by Eck et al. [41] are shown in green and orange symbols above the sequences. The boundaries of these elements differ slightly from that observed in the Grb7 SH2 domain. Residue number is for the Grb7 SH2 domain (b) Cartoon representation of the Grb7 SH2 domain shaded from purple at the N-terminus to red at the C-terminus. The extended DE loop distinguishes this family of SH2 domains from others. (c) A structural comparison of the Grb7 SH2 domain (green) with the Grb7 SH2 domain bound to an ErbB2 derived phosphopeptide (1MW4; black; [29]). The location of the bound phosphopeptide is indicated.
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Figure 1: Crystal structure of the Grb7 SH2 domain. (a) Structure based sequence alignment of Grb7 SH2, Grb14 SH2 (2AUG; [37]) and Grb10 SH2 (1NRV; [36]). Secondary structure elements present in the Grb7 SH2 structure as determined by WHATIF [71] are shaded from purple at the N-terminus to red at the C-terminus. Secondary structure elements of the canonical SH2 domain as defined by Eck et al. [41] are shown in green and orange symbols above the sequences. The boundaries of these elements differ slightly from that observed in the Grb7 SH2 domain. Residue number is for the Grb7 SH2 domain (b) Cartoon representation of the Grb7 SH2 domain shaded from purple at the N-terminus to red at the C-terminus. The extended DE loop distinguishes this family of SH2 domains from others. (c) A structural comparison of the Grb7 SH2 domain (green) with the Grb7 SH2 domain bound to an ErbB2 derived phosphopeptide (1MW4; black; [29]). The location of the bound phosphopeptide is indicated.

Mentions: The monomer fold of the Grb7 SH2 domain is typical of the SH2 domain family, forming a βαβββββαβ configuration [40] (Figure 1). The standard nomenclature for SH2 domain secondary structures devised by Eck et al. [41]is adopted throughout this report. Thus the Grb7 SH2 domain comprises a central antiparallel β-sheet, formed by the βB, βC and βD strands, which is sandwiched by the two α-helices, αA and αB. An extension of the βD strand (termed βD'), along with the βE strand, forms a second, smaller anti-parallel β-sheet. An N-terminal helix was observed prior to the start of the SH2 domain and designated the name H1. The βF strand, observed in other SH2 domains, does not form in Grb7.


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)

Crystal structure of the Grb7 SH2 domain. (a) Structure based sequence alignment of Grb7 SH2, Grb14 SH2 (2AUG; [37]) and Grb10 SH2 (1NRV; [36]). Secondary structure elements present in the Grb7 SH2 structure as determined by WHATIF [71] are shaded from purple at the N-terminus to red at the C-terminus. Secondary structure elements of the canonical SH2 domain as defined by Eck et al. [41] are shown in green and orange symbols above the sequences. The boundaries of these elements differ slightly from that observed in the Grb7 SH2 domain. Residue number is for the Grb7 SH2 domain (b) Cartoon representation of the Grb7 SH2 domain shaded from purple at the N-terminus to red at the C-terminus. The extended DE loop distinguishes this family of SH2 domains from others. (c) A structural comparison of the Grb7 SH2 domain (green) with the Grb7 SH2 domain bound to an ErbB2 derived phosphopeptide (1MW4; black; [29]). The location of the bound phosphopeptide is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Crystal structure of the Grb7 SH2 domain. (a) Structure based sequence alignment of Grb7 SH2, Grb14 SH2 (2AUG; [37]) and Grb10 SH2 (1NRV; [36]). Secondary structure elements present in the Grb7 SH2 structure as determined by WHATIF [71] are shaded from purple at the N-terminus to red at the C-terminus. Secondary structure elements of the canonical SH2 domain as defined by Eck et al. [41] are shown in green and orange symbols above the sequences. The boundaries of these elements differ slightly from that observed in the Grb7 SH2 domain. Residue number is for the Grb7 SH2 domain (b) Cartoon representation of the Grb7 SH2 domain shaded from purple at the N-terminus to red at the C-terminus. The extended DE loop distinguishes this family of SH2 domains from others. (c) A structural comparison of the Grb7 SH2 domain (green) with the Grb7 SH2 domain bound to an ErbB2 derived phosphopeptide (1MW4; black; [29]). The location of the bound phosphopeptide is indicated.
Mentions: The monomer fold of the Grb7 SH2 domain is typical of the SH2 domain family, forming a βαβββββαβ configuration [40] (Figure 1). The standard nomenclature for SH2 domain secondary structures devised by Eck et al. [41]is adopted throughout this report. Thus the Grb7 SH2 domain comprises a central antiparallel β-sheet, formed by the βB, βC and βD strands, which is sandwiched by the two α-helices, αA and αB. An extension of the βD strand (termed βD'), along with the βE strand, forms a second, smaller anti-parallel β-sheet. An N-terminal helix was observed prior to the start of the SH2 domain and designated the name H1. The βF strand, observed in other SH2 domains, does not form in Grb7.

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