Thermodynamic and Structural Analysis of Phosphotyrosine Polypeptide Binding to Grb2-SH2

McNemar, Charles; Snow, Mark E.; Windsor, William; Prongay, Andrew; Mui, Philip W.; Zhang, Rumin; Durkin, James; Le, Hung V.; Weber, Patricia C

doi:10.1021/bi9704360

Cited by 70 publications

(62 citation statements)

References 39 publications

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“…For the linear pYVNV peptide binding to Grb2 proteins, the observed ∆C p value (Table 3) is close to the reported value of -145 (cal/mol)/K. 25 These findings give confidence in the parameters derived from our SPR experiments.…”

Section: Discussionsupporting

confidence: 87%

Surface Plasmon Resonance Thermodynamic and Kinetic Analysis as a Strategic Tool in Drug Design. Distinct Ways for Phosphopeptides to Plug into Src- and Grb2 SH2 Domains

et al. 2005

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Thermodynamic and kinetic studies of biomolecular interactions give insight into specificity of molecular recognition processes and advance rational drug design. Binding of phosphotyrosine (pY)-containing peptides to Src-and Grb2-SH2 domains was investigated using a surface plasmon resonance (SPR)-based method. This SPR assay yielded thermodynamic binding constants in solution, and the kinetic information contained in the SPR signal allowed kinetic analysis, which demonstrated distinct ways for pY ligands to interact with the SH2 domains. The results for binding to Src SH2 were consistent with sequestration of water molecules in the interface of the pYEEI peptide/Src SH2 complex. The results for a pYVNV peptide binding to Grb2 SH2 suggested a conformational change for Grb2 SH2 upon binding, which is not observed for Src SH2. Binding of a cyclic construct, allowing the pYVNV sequence in the bound conformation, did not have the expected entropy advantage. The results suggest an alternative binding mode for this construct, with the hydrophobic ring-closing part interacting with the protein. In all cases, except for full-length Grb2 protein, the affinity for the immobilized peptide at the SPR sensor and in solution was identical. This study demonstrates that SPR thermodynamic and kinetic analysis is a useful strategic tool in drug design.

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Section: Discussionsupporting

confidence: 87%

Surface Plasmon Resonance Thermodynamic and Kinetic Analysis as a Strategic Tool in Drug Design. Distinct Ways for Phosphopeptides to Plug into Src- and Grb2 SH2 Domains

et al. 2005

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“…In our laboratory, we have measured the extinction coefficient of dimer to be 30,120 M −1 , whereas a value of 15,000 M −1 for the pure monomeric form has been measured. Our value for monomer compares reasonably well with that obtained by McNemar and coworkers (15,600 M −1 ) [21].…”

Section: Preparation Of Recombinant Grb2-sh2supporting

confidence: 92%

Structural and energetic aspects of Grb2-SH2 domain-swapping

Benfield

Whiddon

Clements

et al. 2007

Archives of Biochemistry and Biophysics

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The SH2 domain of growth factor receptor-bound protein 2 (Grb2) has been the focus of numerous studies, primarily because of the important roles it plays in signal transduction. More recently, it has emerged as a useful protein to study the consequences of ligand preorganization upon energetics and structure in protein-ligand interactions. The Grb2-SH2 domain is known to form a domain-swapped dimer, and as part of our investigations toward correlating structure and energetics in biological systems, we examined the effects that domain-swapping dimerization of the Grb2-SH2 domain had upon ligand binding affinities. Isothermal titration calorimetry was performed using Grb2-SH2 in both its monomeric and domain-swapped dimeric forms and a phosphorylated tripeptide AcNHpTyr-Val-Asn-NH 2 that is similar to the Shc sequence recognized by Grb2-SH2 in vivo. The two binding sites of domain-swapped dimer exhibited a 4-and a 13-fold reduction in ligand affinity compared to monomer. Crystal structures of peptide-bound and uncomplexed forms of Grb2-SH2 domain-swapped dimer were obtained and reveal that the orientation of residues V122, V123, and R142 may influence the conformation of W121, an amino acid that is believed to play an important role in Grb2-SH2 ligand sequence specificity. These findings suggest that domain-swapping of Grb2-SH2 not only results in a lower affinity for a Shc-derived ligand, but it may also affect ligand specificity.

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“…The binding thermodynamics of Grb2 SH2, p85 nSH2, and p85 cSH2 to phosphopeptides different from the CD28 peptide have been reported previously (21,23,24). The binding affinity of Grb2 SH2 to the Shc-derived phosphopeptide SpYVNVQ is about 10 times higher than that to DdcP02, SDpYMNMT-PRRPG (24).…”

Section: Discussionsupporting

confidence: 51%

“…The binding affinity of Grb2 SH2 to the Shc-derived phosphopeptide SpYVNVQ is about 10 times higher than that to DdcP02, SDpYMNMT-PRRPG (24). The NMR structure of Grb2 SH2 in complex with Shc-derived peptide showed that the Shc peptide forms a ␤-turn (25), which is slightly different from the twisted conformation of CD28-peptide bound to Grb2 SH2.…”

Section: Discussionmentioning

confidence: 98%

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins

Inaba

Numoto

Ogawa

et al. 2017

Journal of Biological Chemistry

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Edited by Norma AllewellFull activation of T cells and differentiation into effector T cells are essential for many immune responses and require costimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptorbound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N-and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases.

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Thermodynamic and Structural Analysis of Phosphotyrosine Polypeptide Binding to Grb2-SH2

Cited by 70 publications

References 39 publications

Surface Plasmon Resonance Thermodynamic and Kinetic Analysis as a Strategic Tool in Drug Design. Distinct Ways for Phosphopeptides to Plug into Src- and Grb2 SH2 Domains

Surface Plasmon Resonance Thermodynamic and Kinetic Analysis as a Strategic Tool in Drug Design. Distinct Ways for Phosphopeptides to Plug into Src- and Grb2 SH2 Domains

Structural and energetic aspects of Grb2-SH2 domain-swapping

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins

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