Purification, stabilization, and crystallization of a modular protein: Grb2

Guilloteau, J.P.; Fromage, Nadine; Riès-Kautt, Madeleine; Reboul, S.; Bocquet, D.; Dubois, H.; Faucher, D.; Colonna, Claudia; Ducruix, A.; Becquart, Jérôme

doi:10.1002/(sici)1097-0134(199605)25:1<112::aid-prot9>3.3.co;2-d

Cited by 20 publications

(14 citation statements)

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“…It is not known whether this aggregation is a direct consequence of dimer formation. Noteworthily, oxidation and subsequent dimer formation have been shown to impair protein crystallization of, for example, Grb2 (58). By replacing C337 with serine in Btk, we succeeded in preventing dimer formation.…”

Section: Discussionmentioning

confidence: 98%

Six X-Linked Agammaglobulinemia-Causing Missense Mutations in the Src Homology 2 Domain of Bruton’s Tyrosine Kinase: Phosphotyrosine-Binding and Circular Dichroism Analysis

Mattsson

Lappalainen

Backesjo

et al. 2000

The Journal of Immunology

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Src homology 2 (SH2) domains recognize phosphotyrosine (pY)-containing sequences and thereby mediate their association to ligands. Bruton’s tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase, in which mutations cause a hereditary immunodeficiency disease, X-linked agammaglobulinemia (XLA). Mutations have been found in all Btk domains, including SH2. We have analyzed the structural and functional effects of six disease-related amino acid substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q. Also, we present a novel Btk SH2 missense mutation, H362R, leading to classical XLA. Based on circular dichroism analysis, the conformation of five of the XLA mutants studied differs from the native Btk SH2 domain, while mutant R307G is structurally identical. The binding of XLA mutation-containing SH2 domains to pY-Sepharose was reduced, varying between 1 and 13% of that for the native SH2 domain. The solubility of all the mutated proteins was remarkably reduced. SH2 domain mutations were divided into three categories: 1) Functional mutations, which affect residues presumably participating directly in pY binding (R307G); 2) structural mutations that, via conformational change, not only impair pY binding, but severely derange the structure of the SH2 domain and possibly interfere with the overall conformation of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural-functional mutations, which contain features from both categories above (Y361C).

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Section: Discussionmentioning

confidence: 98%

Six X-Linked Agammaglobulinemia-Causing Missense Mutations in the Src Homology 2 Domain of Bruton’s Tyrosine Kinase: Phosphotyrosine-Binding and Circular Dichroism Analysis

Mattsson

Lappalainen

Backesjo

et al. 2000

The Journal of Immunology

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“…The final buffer for the gel filtration step was 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 5 mM TCEP. The purification procedure for Grb2 was very similar to that for LAT, except for the final buffer, which included 100 mM arginine to stabilize Grb2 in solution (26).…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for Activation of ZAP-70 by Phosphorylation of the SH2-Kinase Linker

Yan

Barros

Visperas

et al. 2013

Molecular and Cellular Biology

101

141

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Serial activation of the tyrosine kinases Lck and ZAP-70 initiates signaling downstream of the T cell receptor. We previously reported the structure of an autoinhibited ZAP-70 variant in which two regulatory tyrosine residues (315 and 319) in the SH2-kinase linker were replaced by phenylalanine. We now present a crystal structure of ZAP-70 in which Tyr 315 and Tyr 319 are not mutated, leading to the recognition of a five-residue sequence register error in the SH2-kinase linker of the original crystallographic model. The revised model identifies distinct roles for these two tyrosines. As seen in a recently reported structure of the related tyrosine kinase Syk, Tyr 315 of ZAP-70 is part of a hydrophobic interface between the regulatory apparatus and the kinase domain, and the integrity of this interface would be lost upon engagement of doubly phosphorylated peptides by the SH2 domains. Tyr 319 is not necessarily dislodged by SH2 engagement, which activates ZAP-70 only ϳ5-fold in vitro. In contrast, phosphorylation by Lck activates ZAP-70 ϳ100-fold. This difference is due to the ability of Tyr 319 to suppress ZAP-70 activity even when the SH2 domains are dislodged from the kinase domain, providing stringent control of ZAP-70 activity downstream of Lck. Signaling by the T cell receptor (TCR) relies on two nonreceptor tyrosine kinases: the Src family tyrosine kinase Lck and the zeta-chain-associated protein kinase ZAP-70 ( Fig. 1) (1). The clustering of coreceptor (CD4 or CD8)-associated Lck with T cell receptors allows Lck to phosphorylate tyrosine residues in immunoreceptor tyrosine-based activation motifs (ITAMs) in the intracellular tails of zeta chains of the TCR complex. Doubly phosphorylated ITAMs in the stimulated TCR complex recruit ZAP-70 to the plasma membrane, where it is phosphorylated by Lck (2, 3). ITAM binding and phosphorylation release ZAP-70 from an autoinhibited state, enabling it to phosphorylate two scaffold proteins, LAT (linker for the activation of T cells) and SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa), leading to the recruitment of effector proteins that stimulate T cell activation (4, 5).TCR signaling events in a Jurkat-derived ZAP-70-deficient cell line are completely abolished. Hypomorphic ZAP-70 mutant mice, which have partial defects in TCR signaling, develop autoimmunity or autoimmune disease (6-8). The importance of ZAP-70 for T cell development and activation is further emphasized by the fact that the loss of ZAP-70 function results in severe combined immunodeficiency (SCID) in both humans and mice, which is characterized by a lack of functional peripheral T cells (9-12). The tight association of these diseases with ZAP-70 catalytic activity indicates clearly that ZAP-70 is a potential therapeutic target.ZAP-70 and its close relative Syk, which is essential for B cell receptor signaling, share a domain architecture that is unique among protein kinases. The N-terminal half of each protein contains two SH2 domains that form a tightly coupled module in...

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“…The full-length Grb2 protein was expressed and purified as described by Guilloteau et al 44 This protein was in the monomeric form.…”

Section: Methodsmentioning

confidence: 99%

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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Purification, stabilization, and crystallization of a modular protein: Grb2

Cited by 20 publications

References 1 publication

Six X-Linked Agammaglobulinemia-Causing Missense Mutations in the Src Homology 2 Domain of Bruton’s Tyrosine Kinase: Phosphotyrosine-Binding and Circular Dichroism Analysis

Six X-Linked Agammaglobulinemia-Causing Missense Mutations in the Src Homology 2 Domain of Bruton’s Tyrosine Kinase: Phosphotyrosine-Binding and Circular Dichroism Analysis

Structural Basis for Activation of ZAP-70 by Phosphorylation of the SH2-Kinase Linker

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

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