Crystal Structure of the Mammalian Grb2 Adaptor

Maignan, S.; Guilloteau, J.P.; Fromage, Nadine; Arnoux, B.; Becquart, Jérôme; Ducruix, A.

doi:10.1126/science.7716522

Cited by 207 publications

(209 citation statements)

References 28 publications

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“…This observed mobility is reminiscent of the dynamic behavior that has been reported between tandem SH2-SH3 domains of Abl kinase (56,57), Hck kinase (52,58), Lck kinase (59), and Src kinase (60). The recent solution structure of the SH3-SH2-SH3 adapter protein Grb2 also demonstrates a lack of rigid domain orientation (61,62). Functionally, this flexibility may be necessary to facilitate binding to many different independent or consolidated phosphotyrosine-containing and proline-rich ligands from disparate components of the signal transduction machinery.…”

Section: Discussionmentioning

confidence: 80%

Structure of a regulatory complex involving the Abl SH3 domain, the Crk SH2 domain, and a Crk-derived phosphopeptide

Donaldson

Gish

Pawson

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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On phosphorylation of Y221 by Abelson (Abl) kinase, the Crk-II adapter protein undergoes an intramolecular reorganization initiated by the binding of its own Src homology 2 (SH2) domain to the pY221 site. Conformational changes induced by phosphotyrosine recognition promote the binding of the Src homology 3 (SH3) domain of the Abl tyrosine kinase to a proline-rich loop located between the ␤D and ␤E strands of the SH2 domain (DE loop). We have determined the NMR solution structure of the ternary complex of the Abl SH3 domain with the Crk SH2 domain bound to a Crk pY221 phosphopeptide. The SH2 domain bridges two ligands that bind at distinct sites. The interaction between the Abl SH3 domain and the Crk SH2 domain is localized to a canonical eightresidue site within the DE loop. From 15 N relaxation experiments, the DE loop of the SH2 domain in the complex displays a significant degree of conformational freedom. The structural and dynamic data therefore indicate that these SH2 and SH3 domains do not assume a unique orientation with respect to one another; rather, they appear to be only tethered via the DE loop. Thus, SH2 domain-SH3 domain interactions do not require additional tertiary contacts or restriction of domain orientation when a recognition motif is presented in a mobile loop. This complex between the Abl SH3 domain, Crk SH2 domain, and Crk phosphopeptide is an example of the extremely modular nature of regulatory proteins that provides a rich repertoire of mechanisms for control of biological function.NMR ͉ signal transduction ͉ domain orientation ͉ modular binding domain O ur understanding of the regulation of biological processes by protein interactions has been significantly enhanced by the description of modular binding domains and their preferred target sequences (1, 2). In particular, the Src homology 2 (SH2) and Src homology 3 (SH3) domains of cytoplasmic tyrosine kinases and associated adapter proteins demonstrate a wide variety of regulatory mechanisms involving both intramolecular and intermolecular interactions with target peptide regions (3, 4). Whether additional contacts exist beyond those presented by canonical peptide models is an important issue, particularly as they can potentially modulate binding affinity and specificity (5, 6). An interesting example in this context is provided by the protein interactions observed between the Abelson (Abl) tyrosine kinase and the adapter protein Crk-II.Abl has a wide range of functions including signal transduction, cytoskeletal and cell cycle regulation, neural development, and reaction to oxidative stress (7). In addition to its catalytic activity, it possesses an SH2 domain, an SH3 domain, nuclear translocation sequences, a DNA binding domain, and an actin binding domain. Chimeric Abl variants activated as a result of chromosomal translocations (Bcr-Abl, Tel-Abl) or by fusion to viral sequences (v-Abl) are transforming (8, 9) through activation of Ras, phosphoinositol 3-kinase, PKC, and Janus kinase (JAK)͞signal transducers and activators of trans...

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Section: Discussionmentioning

confidence: 80%

Structure of a regulatory complex involving the Abl SH3 domain, the Crk SH2 domain, and a Crk-derived phosphopeptide

Donaldson

Gish

Pawson

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…In the other, the conformation of Arg pB5 is altered such that different Arg pB5 H, protons make contact with the Ser PC5 0, and His PD4 Ngl than shown in Figure 4. Finally, these hydrogen bonds are poorly conserved in the two SH2 domains in the crystallographic dimer observed in the structure of Grb2 (Maignan et al, 1995). In one, the distance between His pD4 HE2 and Glu aA6 0 s atoms is 3.4 A, too far for hydrogen bonding, but the other two hydrogen bonds are present.…”

Section: Histidine Pka Values For the Free Plcc Sh2 Domainmentioning

confidence: 99%

“…These included a refined solution structure of the PLCC SH2 domainpY1021 complex (Pascal et al, 1997) based on the previously published structure (PDB filecode 2PLD; Pascal et al, 1994), as well as crystal structures of the N-terminal SH2 domain of Syp phosphatase (Lee et al, 1994), free (PDB filecode I AYD), and in complex with phosphopeptides (PDB filecodes IAYA, lAYB, and IAYC), the SH2 domain of Grb2 (PDB filecode IGRI; Maignan et al, 1995), the Lck SH2 domain-phosphopeptide complex (PDB filecode ILCJ; Eck et al, 1993) and the Src SH2 domain (Waksman et al, 1993), free (PDB filecode ISPR), and phosphopeptidecomplexed (PDB filecode 1SPS).…”

Section: Methodsmentioning

confidence: 99%

pH Titration studies of an SH2 domain‐phosphopeptide complex: Unusual histidine and phosphate pK_a values

Singer

Forman‐Kay

1997

Protein Science

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Electrostatic interactions in a complex of the phospholipase C-y, C-terminal SH2 domain with a high-affinity binding phosphopeptide representing the sequence around Tyr 1021 of the p platelet-derived growth factor receptor were studied by pK<, determination of various titratable groups over the pH range of 5 to 8. A histidine residue that is highly conserved among SH2 domains (His PD4) and the phosphotyrosine (pTyrj phosphate group show pK', values significantly lower than average for these residue types in proteins. The reduced pK, of these two groups is due to the proximity of the highly positively charged pTyr binding pocket. The unusual pK, of His PD4 is also due to burial from solvent in a hydrogen-bonding network that appears necessary for the positioning of arginine residues involved in pTyr binding. Mutation of the analogous histidine in other SH2 domains has been shown to abrogate pTyr binding. In addition to these large shifts in pK, values, smaller effects were observed for the titratable groups of a glutamic acid and histidine near the C-terminus of the the second helix due to its helical dipole. Finally, exchange behavior of arginine guanidinium protons with solvent as a function of pH in this SH2 domain-phosphopeptide complex confirms previous descriptions of the roles of different arginines in the structure and function of this protein.Keywords: histidine; NMR; phospholipase C-y; phosphotyrosine; pK, determination; SH2 domain Electrostatic interactions in proteins are very important in catalysis, ligand recognition processes, and protein stability (Gilson, 1995). We are interested in the electrostatic interactions of Src homology 2 (SH2) domains with their phosphotyrosine-containing targets. SH2 domains are small ("100 amino acid) domains that play an important role in signal transduction by binding to specific sequences on growth factor receptors or other molecules containing phosphotyrosine (for a review, see Schaffhausen, 1995). Studies of the binding of SH2 domains to their target proteins have been facilitated by modeling of the interacting region of the target protein with small (-20 amino acids or less) phosphopeptides containing sequences around the site of Tyr phosphorylation. These peptides can significantly inhibit the biological interaction (Yonezawa et al., 1992;Larose et al., 1993;Nishimura et al., 1993). SH2 domain binding to phosphopeptides is characterized by a very rapid (diffusion-limited or faster) on-rate (Panayotou et al., 1993), most likely due to electrostatic attraction of the phosphotyrosine (pTyr) phosphate groups with the highly positively charged binding pockets observed in the structures of SH2 domains and SH2 domain-phosphopeptide complexes solved to date (Eck et al., 1993;Waksman et al., 1993;Gilmer

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“…This analysis, together with predictions based on known SH2 domain structures, 21 suggested that the domain comprises residues 1251-1349. Crystallization trials with the corresponding recombinant domain from S. cerevisiae, Schizosaccharomyces pombe, Candida glabrata, and Homo sapiens resulted in crystals for the C. glabrata SH2 domain, 29 and alignment of amino acid sequences of the Spt6 SH2 domains of C. glabrata (C.g.…”

Section: Structure Determination Of the Spt6 Sh2 Domainmentioning

confidence: 99%

Structure and in Vivo Requirement of the Yeast Spt6 SH2 Domain

Dengl

Mayer

Sun

et al. 2009

Journal of Molecular Biology

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During transcription elongation through chromatin, the Ser2-phosphorylated C-terminal repeat domain of RNA polymerase II binds the C-terminal Src homology 2 (SH2) domain of the nucleosome re-assembly factor Spt6. This SH2 domain is unusual in its specificity to bind phosphoserine, rather than phosphotyrosine and because it is the only SH2 domain in the yeast genome. Here, we report the high-resolution crystal structure of the SH2 domain from Candida glabrata Spt6. The structure combines features from both structural subfamilies of SH2 domains, suggesting it resembles a common ancestor of all SH2 domains. Two conserved surface pockets deviate from those of canonical SH2 domains, and may explain the unusual phosphoserine specificity. Differential gene expression analysis reveals that the SH2 domain is required for normal expression of a subset of yeast genes, and is consistent with multiple functions of Spt6 in chromatin transcription.

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Crystal Structure of the Mammalian Grb2 Adaptor

Cited by 207 publications

References 28 publications

Structure of a regulatory complex involving the Abl SH3 domain, the Crk SH2 domain, and a Crk-derived phosphopeptide

Structure of a regulatory complex involving the Abl SH3 domain, the Crk SH2 domain, and a Crk-derived phosphopeptide

pH Titration studies of an SH2 domain‐phosphopeptide complex: Unusual histidine and phosphate pK_a values

Structure and in Vivo Requirement of the Yeast Spt6 SH2 Domain

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Crystal Structure of the Mammalian Grb2 Adaptor

Cited by 207 publications

References 28 publications

Structure of a regulatory complex involving the Abl SH3 domain, the Crk SH2 domain, and a Crk-derived phosphopeptide

Structure of a regulatory complex involving the Abl SH3 domain, the Crk SH2 domain, and a Crk-derived phosphopeptide

pH Titration studies of an SH2 domain‐phosphopeptide complex: Unusual histidine and phosphate pKa values

Structure and in Vivo Requirement of the Yeast Spt6 SH2 Domain

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Product

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pH Titration studies of an SH2 domain‐phosphopeptide complex: Unusual histidine and phosphate pK_a values