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>3.0.co;2-l

Cited by 10 publications

(2 citation statements)

References 27 publications

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“…We generated a model of SH2/SH2 domainswapped full-length GRB2 dimer by combining an SH2/SH2 domain-swapped dimer consisting of the isolated SH2 domain (PDB: 6ICH) and the N-and C-SH3 domains derived from the full-length GRB2 crystal structure (PDB: 1GRI) (Fig. 1D) 60,70,71 . In contrast to the crystal structure, fitting of the full-length SH2/SH2 domainswapped GRB2 dimer to the WT, N188D/N214D, and V122P/V123P dimers gave χ 2 values of 2.2, 16.8, and 6.7, indicating vastly improved fits (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Fractions containing GRB2 were pooled and concentrated using Amicon Ultra-15 centrifugal filter units (MWCO 10 kDa) (Millipore). Particular care is required for sample preparation from this point on, given the increasingly unfavorable conditions (e.g., dialysis for the removal of L-arginine following SEC) and thus, increasing potential for precipitation, as has been reported 64,70 . The protein was further purified by SEC and the concentrate injected onto a 120 mL Superdex 75 10/300 preparative-grade gel filtration column (GE) equilibrated with 20 mM Tris pH 8.0, 150 mM NaCl, 100 mM L-arginine, and 1 mM DTT on a Bio-Rad BioLogic DuoFlow chromatography system.…”

Section: Recombinant Protein Expression Immobilized Metal Ion Affinit...mentioning

confidence: 99%

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GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production

Sandouk

Baruah

et al. 2023

Sci Rep

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GRB2 is an adaptor protein required for facilitating cytoplasmic signaling complexes from a wide array of binding partners. GRB2 has been reported to exist in either a monomeric or dimeric state in crystal and solution. GRB2 dimers are formed by the exchange of protein segments between domains, otherwise known as “domain-swapping”. Swapping has been described between SH2 and C-terminal SH3 domains in the full-length structure of GRB2 (SH2/C–SH3 domain-swapped dimer), as well as between α-helixes in isolated GRB2 SH2 domains (SH2/SH2 domain-swapped dimer). Interestingly, SH2/SH2 domain-swapping has not been observed within the full-length protein, nor have the functional influences of this novel oligomeric conformation been explored. We herein generated a model of full-length GRB2 dimer with an SH2/SH2 domain-swapped conformation supported by in-line SEC–MALS–SAXS analyses. This conformation is consistent with the previously reported truncated GRB2 SH2/SH2 domain-swapped dimer but different from the previously reported, full-length SH2/C-terminal SH3 (C–SH3) domain-swapped dimer. Our model is also validated by several novel full-length GRB2 mutants that favor either a monomeric or a dimeric state through mutations within the SH2 domain that abrogate or promote SH2/SH2 domain-swapping. GRB2 knockdown and re-expression of selected monomeric and dimeric mutants in a T cell lymphoma cell line led to notable defects in clustering of the adaptor protein LAT and IL-2 release in response to TCR stimulation. These results mirrored similarly-impaired IL-2 release in GRB2-deficient cells. These studies show that a novel dimeric GRB2 conformation with domain-swapping between SH2 domains and monomer/dimer transitions are critical for GRB2 to facilitate early signaling complexes in human T cells.

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Section: Resultsmentioning

confidence: 99%

Section: Recombinant Protein Expression Immobilized Metal Ion Affinit...mentioning

confidence: 99%

GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production

Sandouk

Baruah

et al. 2023

Sci Rep

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Principles of Macromolecular X‐Ray Crystallography

Hickman

Davies

1997

CP Protein Science

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Identification of Polyproline II Regions Derived From the Proline‐Rich Nuclear Receptor Coactivators PNRC and PNRC2: New Insights for ERα Coactivator Interactions

et al. 2013

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Protein-protein interactions are crucial for signal transductions required for cell differentiation and proliferation. Their modulation is therefore key to the development of therapeutic alternatives, particularly in the context of cancer. According to literature data, the polyproline-rich nuclear receptor coactivators PNRC and PNRC2 interact with estrogen receptor (ERα) through their PxxP SH3-binding motifs. In a search to identify the molecular features governing this interaction, we explored using electronic circular dichroism (ECD) spectroscopy and molecular dynamics (MD) calculations, the capacity of a range of putative biologically active peptides derived from these proteins and containing this PxxP motif(s) to form polyproline II (PPII) domains. An additional more exhaustive structural study on a lead PPII peptide was also performed using 2D nuclear magnetic resonance (NMR) spectroscopy. With the exception of one of all the investigated peptides (PNRC-D), binding assays failed to detect any affinity for Grb2 SH3 domains, suggesting that PPII motifs issued from Grb2 antagonists have a binding mode distinct from those derived from Grb2 agonists. Instead, the peptides revealed a competitive binding ability against a synthetic peptide (ERα17p) with a putative PPII-cognate domain located within a coregulator recruitment region of ERα (AF-2 site). Our work, which constitutes the first structure-related interaction study concerning PNRC and PNRC2, supports not only the existence of PxxP-induced PPII sequences in these coregulators, but also confirms the presence of a PPII recognition site in the AF-2 of the steroid receptor ERα, a region important for transcription regulation.

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

Cited by 10 publications

References 27 publications

GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production

GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production

Principles of Macromolecular X‐Ray Crystallography

Identification of Polyproline II Regions Derived From the Proline‐Rich Nuclear Receptor Coactivators PNRC and PNRC2: New Insights for ERα Coactivator Interactions

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