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Selectivity and mechanism of action of a growth factor receptor-bound protein 2 SRC homology 2 domain binding antagonist.

ABSTRACT: We have shown previously that a potent synthetic antagonist of growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain binding (1) blocks growth factor stimulated motility, invasion, and angiogenesis in cultured cell models, as well as tumor metastasis in animals. To characterize the selectivity of 1 for the SH2 domain of Grb2 over other proteins containing similar structural binding motifs, we synthesized a biotinylated derivative (3) that retained high affinity Grb2 SH2 domain binding and potent biological activity. To investigate the selectivity of 1 and 3 for Grb2, the biotinylated antagonist 3 was used to immobilize target proteins from cell extracts for subsequent identification by mass spectrometry. Non-specific binding was identified in parallel using a biotinylated analogue that lacked a single critical binding determinant. The mechanism of action of the antagonist was further characterized by immunoprecipitation, immunoblotting, and light microscopy. This approach to defining protein binding antagonist selectivity and molecular basis of action should be widely applicable in drug development.

SUBMITTER: Giubellino A

PROVIDER: S-EPMC2651228 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Selectivity and mechanism of action of a growth factor receptor-bound protein 2 SRC homology 2 domain binding antagonist.

Giubellino Alessio A Shi Zhen-Dan ZD Jenkins Lisa M Miller LM Worthy Karen M KM Bindu Lakshman K LK Athauda Gagani G Peruzzi Benedetta B Fisher Robert J RJ Appella Ettore E Burke Terrence R TR Bottaro Donald P DP

Journal of medicinal chemistry 20081201 23

We have shown previously that a potent synthetic antagonist of growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain binding (1) blocks growth factor stimulated motility, invasion, and angiogenesis in cultured cell models, as well as tumor metastasis in animals. To characterize the selectivity of 1 for the SH2 domain of Grb2 over other proteins containing similar structural binding motifs, we synthesized a biotinylated derivative (3) that retained high affinity Grb2 SH2 domai ...[more]

PMID: 18989951

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Project description:SH2 domain-containing tyrosine phosphatase 2 (SHP2), encoded by PTPN11, plays a fundamental role in the modulation of several signaling pathways. Germline and somatic mutations in PTPN11 are associated with different rare diseases and hematologic malignancies, and recent studies have individuated SHP2 as a central node in oncogenesis and cancer drug resistance. The SHP2 structure includes two Src homology 2 domains (N-SH2 and C-SH2) followed by a catalytic protein tyrosine phosphatase (PTP) domain. Under basal conditions, the N-SH2 domain blocks the active site, inhibiting phosphatase activity. Association of the N-SH2 domain with binding partners containing short amino acid motifs comprising a phosphotyrosine residue (pY) leads to N-SH2/PTP dissociation and SHP2 activation. Considering the relevance of SHP2 in signaling and disease and the central role of the N-SH2 domain in its allosteric regulation mechanism, we performed microsecond-long molecular dynamics (MD) simulations of the N-SH2 domain complexed to 12 different peptides to define the structural and dynamical features determining the binding affinity and specificity of the domain. Phosphopeptide residues at position -2 to +5, with respect to pY, have significant interactions with the SH2 domain. In addition to the strong interaction of the pY residue with its conserved binding pocket, the complex is stabilized hydrophobically by insertion of residues +1, +3, and +5 in an apolar groove of the domain and interaction of residue -2 with both the pY and a protein surface residue. Additional interactions are provided by hydrogen bonds formed by the backbone of residues -1, +1, +2, and +4. Finally, negatively charged residues at positions +2 and +4 are involved in electrostatic interactions with two lysines (Lys89 and Lys91) specific for the SHP2 N-SH2 domain. Interestingly, the MD simulations illustrated a previously undescribed conformational flexibility of the domain, involving the core β sheet and the loop that closes the pY binding pocket.

Dataset Information

Selectivity and mechanism of action of a growth factor receptor-bound protein 2 SRC homology 2 domain binding antagonist.

Publications

Selectivity and mechanism of action of a growth factor receptor-bound protein 2 SRC homology 2 domain binding antagonist.

Similar Datasets

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