Structural and energetic aspects of Grb2-SH2 domain-swapping

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 AcNH-pTyr-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.     

Calorimetric investigation of phosphorylated and non-phosphorylated peptide ligand binding to the human Grb7-SH2 domain

Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20-30% of breast cancers. In general, growth factor receptor bound (Grb) proteins bind to activated membrane-bound receptor tyrosine kinases (RTKs; e.g., the epidermal growth factor receptor, EGFR) through their Src homology 2 (SH2) domains. In particular, Grb7 binds to erbB2 (a.k.a. EGFR2) and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In previous studies, we reported the solution structure and the backbone relaxation behavior of the Grb7-SH2/erbB2 peptide complex. In this study, isothermal titration calorimetry studies have been completed by measuring the thermodynamic binding parameters of several phosphorylated and non-phosphorylated peptides representative of natural Grb7 receptor ligands as well as ligands developed through combinatorial peptide screening methods. The entirety of these calorimetric studies is interpreted in an effort to describe the specific ligand binding characteristics of the Grb7 protein.     

用Gateway 系统构建携带Grb2-SH2 基因重组慢病毒载体

目的:构建携带Grb2-SH2基因重组慢病毒载体。方法:把Grb2-SH2基因从合成的工程载体PUC-57中导到入门载体pentr-3C中,再利用LR反应重组到目的载体plenti,通过酶切和测序分析对比验证Grb2-SH2基因后,将plenti-Grb2-SH2质粒利用慢病毒转染系统转染人胚胎肾上皮细胞株293T细胞获得携带Grb2-SH2慢病毒载体。结果:构建的重组质粒经酶切及测序和分析比对鉴定正确,目的基因片段大小约为500bp;该质粒与包装质粒共转染293T细胞获取的慢病毒,转染效率约为90%。结论:成功构建转载质粒plenti-Grb2-SH2及携带Grb2-SH2基因慢病毒载体。     

Solution structure of N-terminal SH3 domain of Vav and the recognition site for Grb2 C-terminal SH3 domain

The three-dimensional structure of the N-terminal SH3 domain (residues 583–660) of murine Vav, which contains a tetra-proline sequence (Pro 607-Pro 610), was determined by NMR. The solution structure of the SH3 domain shows a typical SH3 fold, but it exists in two conformations due to cis-trans isomerization at the Gly614-Pro615 bond. The NMR structure of the P615G mutant, where Pro615 is replaced by glycine, reveals that the tetra-proline region is inserted into the RT-loop and binds to its own SH3 structure. The C-terminal SH3 domain of Grb2 specifically binds to the trans form of the N-terminal SH3 domain of Vav. The surface of Vav N-terminal SH3 which binds to Grb2 C-terminal SH3 was elucidated by chemical shift mapping experiments using NMR. The surface does not involve the tetra-proline region but involves the region comprising the n-src loop, the N-terminal and the C-terminal regions. This surface is located opposite to the tetra-proline containing region, consistent with that of our previous mutagenesis studies.     

Interaction of the non-phosphorylated peptide G7-18NATE with Grb7-SH2 domain requires phosphate for enhanced affinity and specificity

Src‐homology (SH2) domains are an attractive target for the inhibition of specific signalling pathways but pose the challenge of developing a truly specific inhibitor. The G7‐18NATE cyclic peptide is reported to specifically inhibit the growth factor receptor bound protein 7 (Grb7) adapter protein, implicated in the progression of several cancer types, via interactions with its SH2 domain. G7‐18NATE effectively inhibits the interaction of Grb7 with ErbB3 and focal adhesion kinase in cell lysates and, with the addition of a cell permeability sequence, inhibits the growth and migration of a number of breast cancer cell lines. It is thus a promising lead in the development of therapeutics targeted to Grb7. Here we investigate the degree to which G7‐18NATE is specific for the Grb7‐SH2 domain compared with closely related SH2 domains including those of Grb10, Grb14, and Grb2 using surface plasmon resonance. We demonstrate that G7‐18NATE binds with micromolar binding affinity to Grb7‐SH2 domain (K_D = 4–6 μm ) compared with 50–200 times lower affinity for Grb10, Grb14, and Grb2 but that this specificity depends critically on the presence of phosphate in millimolar concentrations. Other differences in buffer composition, including use of Tris or 2‐(N‐Morpholino)ethanesulfonic acid or varying the pH, do not impact on the interaction. This suggests that under cellular conditions, G7‐18NATE binds with highest affinity to Grb7. In addition, our findings demonstrate that the basis of specificity of G7‐18NATE binding to the Grb7‐SH2 domain is via other than intrinsic structural features of the protein, representing an unexpected mode of molecular recognition. Copyright © 2011 John Wiley & Sons, Ltd.     

Structure-based design of potent Grb2-SH2 domain antagonists not relying on phosphotyrosine mimics

Development of Grb2-SH2 domain antagonists is considered to be an effective and non-cytotoxic strategy to develop new antiproliferative agents because of their potential to shut down the Ras signaling pathway. We developed a concise route for the efficient synthesis of G1TE analogs on solid phase. Using this route, a series of cyclic peptides that do not rely on phosphotyrosine or its mimics were designed and synthesized based upon the phage library-derived cyclopeptide, G1TE. Considering that Gly7 plays prominent roles for G1TE binding to the Grb2-SH2 domain, we introduced different amino acids in the 7th position. The D-Ala7-containing peptide 3 demonstrates improved binding affinity by adopting favorable conformation for protein binding. This can be rationalized by molecular modeling. The optimization at the Leu2 position was also studied, and the resulting cyclopeptides exhibited remarkably improved binding affinity. Based upon these global modifications, a highly potent peptide ligand 9 was discovered with a Kd = 17 nM, evaluated by Biacore binding assay. This new analog is one of the most potent non-phosphorus-containing Grb2-SH2 antagonists reported to date. This potent peptidomimetic provides a new template for the development of non-pTyr containing Grb2-SH2 domain antagonists and acts as a chemotherapeutic lead for the treatment of erbB2-related cancer.     

Conformation of an Shc-derived phosphotyrosine-containing peptide complexed with the Grb2 SH2 domain

We have determined the structure of an Shc-derivedphosphotyrosine-containing peptide complexed with Grb2 SH2 based on intra-and intermolecular NOE correlations observed by a series of isotope-filteredNMR experiments using a PFG z-filter. In contrast to an extendedconformation of phosphotyrosine-containing peptides bound to Src, Syp andPLC SH2s, the Shc-derived peptide formed a turn at the +1 and +2positions next to the phosphotyrosine residue. Trp¹²¹, locatedat the EF1 site of Grb2 SH2, blocked the peptide binding in an extendedconformation. The present study confirms that eachphosphotyrosine-containing peptide binds to the cognate SH2 with a specificconformation, which gives the structural basis for the binding specificitybetween SH2s and target proteins.     

Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor

The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity.     

Chemical shift assignments and secondary structure of the Grb2 SH2 domain by heteronuclear NMR spectroscopy

Summary The growth factor receptor-bound protein-2 (Grb2) is an adaptor protein that mediates signal transduction pathways. Chemical shift assignments were obtained for the SH2 domain of Grb2 by heteronuclear NMR spectroscopy, employing the uniformly ¹³C-/¹⁵N-enriched protein as well as the protein containing selectively ¹⁵N-enriched amino acids. Using the Chemical Shift Index (CSI) method, the chemical shift indices of four nuclei, ¹H, ¹³C, ¹³C and ¹³CO, were used to derive the secondary structure of the protein. Nuclear Overhauser enhancements (NOEs) were then employed to confirm the secondary structure. The CSI results were compared to the secondary structural elements predicted for the Grb2 SH2 domain from a sequence alignment [Lee et al. (1994) Structure, 2, 423–438]. The core structure of the SH2 domain contains an antiparallel -sheet and two -helices. In general, the secondary structural elements determined from the CSI method agree well with those predicted from the sequence alignment.Abbreviations crk viral p47^gag-crk - EGF epidermal growth factor - GAP GTPase-activating protein - PI3K phosphatidylinositol-3-kinase - PLC- phospholipase-C-, shc, src homologous and collagen - src sarcoma family of nonreceptor tyrosine kinase     

Development of Grb2 SH2 Domain Signaling Antagonists: A Potential New Class of Antiproliferative Agents

Aberrant signaling through protein-tyrosine kinase (PTK)-dependent pathways is associated with several proliferative diseases. Accordingly, PTK inhibitors are being developed as new approaches for the treatment of certain cancers. Growth factor receptor bound protein 2 (Grb2) is an important downstream mediator of PTK signaling that serves obligatory roles in many pathogenic processes. One of the primary functions of Grb2 is to bind to specific phosphotyrosyl (pTyr)-containing sequences through its Src homology 2 (SH2) domain. Agents that bind to the Grb2 SH2 domain and prevent its normal function could disrupt associated PTK signaling and serve as alternatives to kinase-directed inhibitors. Starting from the X-ray crystal structure of a lead peptide bound to the Grb2 SH2 domain, this review will summarize important contributions to these efforts. The presentation will be thematically arranged according to the region of peptide modified, proceeding from the N-terminus to the C-terminus, with a special section devoted to aspects of conformational constraint.     

Grb2 dominantly associates with dynamin II in human hepatocellular carcinoma HepG2 cells

The two SH3 domains and one SH2 domain containing adaptor protein Grb2 is an essential element of the Ras signaling pathway in multiple systems. The SH2 domain of Grb2 recognizes and interacts with phosphotyrosine residues on activated tyrosine kinases, whereas the SH3 domains bind to several proline‐rich domain‐containing proteins such as Sos1. To define the difference in Grb2‐associated proteins in hepatocarcinoma cells, we performed coprecipitation analysis using recombinant GST‐Grb2 fusion proteins and found that several protein components (p170, p125, p100, and p80) differently associated with GST‐Grb2 proteins in human Chang liver and hepatocarcinoma HepG2 cells. Sos1 and p80 proteins dominantly bind to Grb2 fusion proteins in Chang liver, whereas p100 remarkably associate with Grb2 in HepG2 cells. Also GST‐Grb2 SH2 proteins exclusively bound to the p46^Shc, p52^Shc, and p66^Shc are important adaptors of the Ras pathway in HepG2 cells. The p100 protein has been identified as dynamin II. We observed that the N‐SH3 and C‐SH3 domains of Grb2 fusion proteins coprecipitated with dynamin II besides Sos1. These results suggest that dynamin II may be a functional molecule involved in Grb2‐mediated signaling pathway on Ras activation for tumor progression and differentiation of hepatocarcinoma cells. J. Cell. Biochem. 84: 150–155, 2002. © 2001 Wiley‐Liss, Inc.     

Crystal structures of a high-affinity macrocyclic peptide mimetic in complex with the Grb2 SH2 domain

The high-affinity binding of the growth factor receptor-bound protein 2 (Grb2) SH2 domain to tyrosine-phosphorylated cytosolic domains of receptor tyrosine kinases (RTKs) is an attractive target for therapeutic intervention in many types of cancer. We report here two crystal forms of a complex between the Grb2 SH2 domain and a potent non-phosphorus-containing macrocyclic peptide mimetic that exhibits significant anti-proliferative effects against erbB-2-dependent breast cancers. This agent represents a "second generation" inhibitor with greatly improved binding affinity and bio-availability compared to its open-chain counterpart. The structures were determined at 2.0A and 1.8A with one and two domain-swapped dimers per asymmetric unit, respectively. The mode of binding and specific interactions between the protein and the inhibitor provide insight into the high potency of this class of macrocylic compounds and may aid in further optimization as part of the iterative rational drug design process.     

Functional implications of the conformational switch in AICD peptide upon binding to Grb2-SH2 domain

It has been hypothesized previously that synergistic effect of both amyloid precursor protein intracellular C-terminal domain (AICD) and Aβ aggregation could contribute to Alzheimer's disease pathogenesis. Structural studies of AICD have found no stable globular fold over a broad range of pH. Present work is based on the premises that a conformational switch involving the flipping of C-terminal helix of AICD would be essential for effective binding with the Src homology 2 (SH2) domain of growth factor receptor binding protein-2 (Grb2) and subsequent initiation of Grb2-mediated endo-lysosomal pathway. High-resolution crystal structures of Grb2-SH2 domain bound to AICD peptides reveal a unique mode of binding where the peptides assume a noncanonical conformation that is unlike other structures of AICD peptides bound to protein-tyrosine-binding domains or that of its free state; rather, a flipping of the C-terminal helix of AICD is evident. The involvement of different AICD residues in Grb2-SH2 interaction is further elucidated through fluorescence-based assays. Our results reveal the significance of a specific interaction of the two molecules to optimize the rapid transport of AICD inside endosomal vesicles presumably to reduce the cytotoxic load.     

Binding affinity difference induced by the stereochemistry of the sulfoxide bridge of the cyclic peptide inhibitors of Grb2-SH2 domain: NMR studies for the structural origin

The SAR study on a phage library-derived non-phosphorylated cyclic peptide ligand of Grb2-SH2 domain indicates that the configuration of the cyclization linkage is crucial for assuming the active binding conformation. When the thioether linkage was oxidized to the two chiral sulfoxides, the R-configured sulfoxide-cyclized peptide displayed 10-30 times more potency than the corresponding S-configured one in binding affinity to the Grb2-SH2 domain. In this paper, the solution structures of such a pair of sulfoxide-bridged cyclic peptide diastereoisomers, i.e., cyclo[CH(2)CO-Gla(1)-L-Y-E-N-V-G-NPG-Y-(R/S)C(O)(10)]-amide, were determined by NMR and molecular dynamics simulation. Results indicate that the consensus sequence of Y(3)-E(4)-N(5)-V(6) in both diastereoisomers adopt a beta-turn conformation; however, the R-configured peptide forms an extended structure with a circular backbone conformation, while the S-configured isomer forms a compact structure with key residues buried inside the molecule. The average root-mean-square deviations were found to be 0.756 and 0.804 A, respectively. It is apparent that the chiral S-->O group played a key role in the solution structures of the sulfoxide-bridged cyclic peptides. The R-sulfoxide group forms an intramolecular hydrogen bond with the C-terminal amide, conferring a more rigid conformation with all residues protruding outside except for Leu2, in which the Gla1 and Tyr3 share an overlapping function as previous SAR studies proposed. Additionally, the extended structure endows a more hydrophilic binding surface of the R-configured peptide to facilitate its capture by its targeted protein. In comparison, the S-configured sulfoxide was embedded inside the ligand peptide leading to a compact structure, in which the essential residues of Gla1, Tyr3, and Asn5 form multiple intramolecular hydrogen bonds resulting in an unfavorable conformational change and a substantial loss of the interaction with the protein. The solution structures disclosed by our NMR and molecular dynamics simulation studies provide a molecular basis for understanding how the chirality of the cyclization linkage remarkably discriminates in terms of the binding affinity, thus advancing the rational design of potent non-phosphorylated inhibitors of Grb2-SH2 domain as antitumor agents.     

Potentiating effect of distant sites in non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domain

Without the presence of a phosphotyrosyl group, a phage library derived non-phosphorylated cyclic peptide ligand of Grb2-SH2 domain attributed its high affinity and specificity to well-defined and highly favored interactions of its structural elements with the binding pocket of the protein. We have disclosed a significant compensatory role of the Glu(2-) sidechain for the absence of the phosphate functionality on Tyr(0) in the peptide ligand, cyclo(CH(2)CO-Glu(2-)-Leu-Tyr(0)-Glu-Asn-Val-Gly-Met(5+)-Tyr-Cys)-amide (termed G1TE). In this study, we report the importance of hydrophobic residue at the Tyr+5 site in G1TE. Both acidic and basic amino acid substitutes are disfavored at this position, and replacement of Met with beta-tert-butyl-Ala was found to improve the antagonist properties. Besides, the polarity of the cyclization linkage was implicated as important in stabilizing the favored binding conformation. Oxidation of the thioether linkage into sulfoxide facilitated the binding to Grb2-SH2 markedly. Simultaneous modification of the three distant sites within G1TE provided the best agent with an IC(50) of 220 nM, which is among the most potent non-phosphorous- and non-phosphotyrosine-mimic containing Grb2-SH2 domain inhibitors yet reported. This potent peptidomimetic provides a novel template for the development of chemotherapeutic agents for the treatment of erbB2-related cancer. Biological assays on G1TE(Gla(2-)) in which the original residue of Glu(2-) was substituted by gamma-carboxyglutamic acid (Gla) indicated that it could inhibit the interaction between activated GF receptor and Grb2 protein in cell homogenates of MDA-MB-453 breast cancer cells at the 2 microM level. More significantly, both G1TE(Gla(2-)) alone and the conjugate of G1TE(Gla(2-)) with a peptide carrier can effectively inhibit intracellular association of erbB2 and Grb2 in the same cell lines with IC(50) of 50 and 2 microM, respectively.     

Solution structure of the human Grb14-SH2 domain and comparison with the structures of the human Grb7-SH2/erbB2 peptide complex and human Grb10-SH2 domain

Grb14 is an adapter protein that is known to be overexpressed in estrogen receptor positive breast cancers, and in a number of prostate cancer cell lines. Grb14 has been demonstrated to bind to a number of activated receptor tyrosine kinases (RTKs) and to modulate signals transduced through these receptors. The RTKs to which Grb14 binds include the insulin receptor (IR), the fibroblast growth factor receptor (FGFR), the platelet-derived growth factor receptor (PDGFR), and the tunica endothelial kinase (Tek/Tie2) receptor. Grb14 has been shown to bind to these activated RTKs through its Src homology 2 (SH2) domain, with the exception of the insulin receptor, where the primary binding interaction is via a small domain adjacent to the SH2 domain (the BPS or PIR domain). Grb14 is a member of the Grb7 family of proteins, which also includes Grb7 and Grb10. We have solved the solution structure of the human Grb14-SH2 domain and compared it with the recently determined Grb7-SH2 and Grb10-SH2 domain structures.     

Grb7-SH2 domain dimerisation is affected by a single point mutation

Growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is co-overexpressed and forms a tight complex with the ErbB2 receptor in a number of breast tumours and breast cancer cell lines. The interaction of Grb7 with the ErbB2 receptor is mediated via its Src homology 2 (SH2) domain. Whilst most SH2 domains exist as monomers, recently reported studies have suggested that the Grb7-SH2 domain exists as a homodimer. The self-association properties of the Grb7-SH2 domain were therefore studied using sedimentation equilibrium ultracentrifugation. Analysis of the data demonstrated that the Grb7-SH2 domain is dimeric with a dissociation constant of approximately 11 M. We also demonstrate, using size-exclusion chromatography, that mutation of phenylalanine 511 to an arginine produces a monomeric form of the Grb7-SH2 domain. This mutation represents the first step in the engineering of a Grb7-SH2 domain with good solution properties for further biophysical and structural investigation.     

Grb7 protein RA domain oligomerization

The growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is often coamplified with the erythroblastosis oncogene B 2 receptor in 20% to 30% of breast cancer patients. Grb7 overexpression has been linked to increased cell migration and cancer metastasis. The ras associating and pleckstrin homology domain region of Grb7 has been reported to interact with various other downstream signaling proteins such as four and half Lin11, Isl‐1, Mec‐3 (LIM) domains isoform 2 and filamin α. These interactions are believed to play a role in regulating Grb7‐mediated cell migration function. The full‐length Grb7 protein has been shown to dimerize, and the oligomeric state of the Grb7SH2 domain has been extensively studied; however, the oligomerization state of the ras associating and pleckstrin homology domains, and the importance of this oligomerization in Grb7 function, is yet to be fully known. In this study, we characterize the oligomeric state of the Grb7RA domain using size exclusion chromatography, nuclear magnetic resonance, nuclear relaxation studies, glutaraldehyde cross linking, and dynamic light scattering. We report the Grb7RA domain can exist in transient multimeric forms and, based upon modeling results, postulate the potential role of Grb7RA domain oligomerization in Grb7 function.     

Affinity chromatography for purification of the modular protein growth factor receptor-bound protein 2 and development of a screening test for growth factor receptor-bound protein 2 Src homology 3 domain inhibitor using peroxidase-linked ligand

Growth factor receptor-bound protein 2 (Grb2) is an adapter protein involved in the Ras-dependent signaling pathway that plays an important role in human cancers initiated by oncogenic receptors. Grb2 is constituted by one Src homology 2 domain surrounded by two SH3 domains, and the inhibition of the interactions produced by these domains could provide an antitumor approach. In evaluating chemical libraries, to search for potential Grb2 inhibitors, it was necessary to elaborate a rapid test for their screening. We have developed, first, a batch method based on the use of an affinity column bearing a Grb2-SH3 peptide ligand to isolate highly purified Grb2. We subsequently describe a very rapid 96-well screening of inhibitors based on a simple competition between purified Grb2 and a peroxidase-coupled proline-rich peptide.