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ABSTRACT: The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here we report the structural analysis of both holo- and apo- forms of SaBPL using X-ray crystallography. We also present small angle X-ray scattering data of SaBPL in complex with its biotin carboxyl carrier protein (BCCP) substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5'-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery.
Protein Science 04/2013; · 2.80 Impact Factor
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ABSTRACT: The RNA-binding protein TIAR is an mRNA-binding protein that acts as a translational repressor, particularly important under conditions of cellular stress. It binds to target mRNA and DNA via its RNA recognition motif (RRM) domains and is involved in both splicing regulation and translational repression via the formation of "stress granules." TIAR has also been shown to bind ssDNA and play a role in the regulation of transcription. Here we show, using surface plasmon resonance and nuclear magnetic resonance spectroscopy, specific roles of individual TIAR domains for high-affinity binding to RNA and DNA targets. We confirm that RRM2 of TIAR is the major RNA- and DNA-binding domain. However, the strong nanomolar affinity binding to U-rich RNA and T-rich DNA depends on the presence of the six amino acid residues found in the linker region C-terminal to RRM2. On its own, RRM1 shows preferred binding to DNA over RNA. We further characterize the interaction between RRM2 with the C-terminal extension and an AU-rich target RNA sequence using NMR spectroscopy to identify the amino acid residues involved in binding. We demonstrate that TIAR RRM2, together with its C-terminal extension, is the major contributor for the high-affinity (nM) interactions of TIAR with target RNA sequences.
RNA biology 04/2013; 10(4). · 5.56 Impact Factor
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ABSTRACT: Grb7 is a non-catalytic protein, the overexpression of which has been associated with the proliferative and migratory potentials of cancer cells. Virtual screening strategies involving a shape-based similarity search, molecular docking, and 2D-similarity searches complemented by experimental binding studies (Thermofluor and isothermal titration calorimetry) resulted in the identification of nine novel phenylbenzamide-based antagonists of the Grb7 SH2 domain. Moderate binding affinities were observed, ranging from K(d) =32.3 μM for lead phenylbenzamide NSC 104999 (1) to K(d) =1.1 μM for a structurally related compound, NSC 57148 (2). Deconvolution of the affinity data into its components revealed differences in lead binding, from being entropy based (lead 1) to enthalpically driven (NSC 100874 (3), NSC 55158 (4), and compound 2). Finally, the lead compound 1 was found to decrease the growth of MDA-MB-468 breast cancer cells, with an IC(50) value of 39.9 μM. It is expected that these structures will serve as novel leads in the development of Grb7-based anticancer therapeutics.
ChemMedChem 02/2013; 8(2):280-8. · 3.15 Impact Factor
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ABSTRACT: Surface plasmon resonance (SPR) is a useful biosensor technique for the study of biomolecular interactions, with the potential
for high-throughput screening of ligand interactions with drug targets. The key to its successful use, however, is in the
appropriate design of the experiment, including the mode of immobilization to the biosensor chip. We report an investigation
of the use of SPR for measuring the affinity of the G7-18NATE peptide ligand for its Grb7-SH2 domain target involved in the
migratory and proliferative potential of cancer cells. Previous studies have shown that the cyclic non-phosphorylated peptide,
G7-18NATE, inhibits Grb7 interactions with upstream binding partners and is able to inhibit both cell migration and proliferation
of cancer cells. We report the synthesis of a biotinylated G7-18NATE covalently attached to a linker (G7-18NATE-ASASASK-Biotin)
and compare its interaction with the Grb7-SH2 domain by SPR using three different immobilization strategies; immobilisation
of the peptide via streptavidin, immobilization of glutathione S-transferase (GST)-Grb7-SH2 domain via anti-GST antibody,
and immobilization of biotinylated Grb7-SH2 domain via streptavidin. This revealed that sensorgrams free from non-specific
binding and displaying simple kinetics were most readily achieved by immobilising the protein rather than the peptide, in
spite of the lower response associated with this method. K
D values of ~300 μM were determined for both strategies at pH 7.4. This compared with a K
D value of 4.4 μM at pH 6 demonstrating the importance of pH on this interaction. Overall, the immobilised protein systems
are most suitable for future comparative screening efforts using SPR.
KeywordsGrb7 adapter protein-SH2 domain-Non-phosphorylated cyclic peptide-Peptide inhibitor-Surface plasmon resonance
International Journal of Peptide Research and Therapeutics 04/2012; 16(3):177-184. · 0.99 Impact Factor
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ABSTRACT: Experimental and theoretical data indicate that, for α-fluoroamides, the F-C-C(O)-N(H) moiety adopts an antiperiplanar conformation. In addition, a gauche conformation is favoured between the vicinal C-F and C-N(CO) bonds in N-β-fluoroethylamides. This study details the synthesis of a series of fluorinated β-peptides (1-8) designed to use these stereoelectronic effects to control the conformation of β-peptide bonds. X-ray crystal structures of these compounds revealed the expected conformations: with fluorine β to a nitrogen adopting a gauche conformation, and fluorine α to a C=O group adopting an antiperiplanar conformation. Thus, the strategic placement of fluorine can control the conformation of a β-peptide bond, with the possibility of directing the secondary structures of β-peptides.
Chemistry 04/2012; 18(21):6655-62. · 5.93 Impact Factor
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Tatiana P Soares da Costa,
William Tieu,
Min Y Yap,
Nicole R Pendini,
Steven W Polyak,
Daniel Sejer Pedersen,
Renato Morona,
John D Turnidge,
John C Wallace, Matthew C J Wilce,
Grant W Booker,
Andrew D Abell
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ABSTRACT: There is a well documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy to combat resistance is to discover new chemical classes immune to current resistance mechanisms that inhibit essential metabolic enzymes. Many of the obvious drug targets that have no homologous isozyme in the human host have now been investigated. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme over the human homolog. We have demonstrated that the essential enzyme biotin protein ligase (BPL) from the clinically important pathogen Staphylococcus aureus could be selectively inhibited. Linking biotin to adenosine via a 1,2,3 triazole yielded the first BPL inhibitor selective for S. aureus BPL over the human equivalent. The synthesis of new biotin 1,2,3-triazole analogues using click chemistry yielded our most potent structure (K(i) 90 nM) with a >1100-fold selectivity for the S. aureus BPL over the human homologue. X-ray crystallography confirmed the mechanism of inhibitor binding. Importantly, the inhibitor showed cytotoxicity against S. aureus but not cultured mammalian cells. The biotin 1,2,3-triazole provides a novel pharmacophore for future medicinal chemistry programs to develop this new antibiotic class.
Journal of Biological Chemistry 03/2012; 287(21):17823-32. · 4.77 Impact Factor
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ABSTRACT: The stability of RNAs bearing AU-rich elements in their 3'-UTRs, and thus the level of expression of their protein products, is regulated by interactions with cytoplasmic RNA-binding proteins. Binding by HuR generally leads to mRNA stabilization and increased protein production, whereas binding by AUF1 isoforms generally lead to rapid degradation of the mRNA and reduced protein production. The exact nature of the interplay between these and other RNA-binding proteins remains unclear, although recent studies have shown close interactions between them and even suggested competition between the two for binding to their cognate recognition sequences. Other recent reports have suggested that the sequences recognized by the two proteins are different. We therefore performed a detailed in vitro analysis of the binding site(s) for HuR and AUF1 present in androgen receptor mRNA to define their exact target sequences, and show that the same sequence is contacted by both proteins. Furthermore, we analysed a proposed HuR target within the 3'-UTR of MTA1 mRNA, and show that the contacted bases lie outside of the postulated motif and are a better match to a classical ARE than the postulated motif. The defining features of these HuR binding sites are their U-richness and single strandedness.
Journal of biochemistry 02/2012; 151(4):423-37. · 1.95 Impact Factor
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ABSTRACT: Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.
Nucleic Acids Research 02/2012; 40(11):5101-14. · 8.03 Impact Factor
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ABSTRACT: A library of 14-helical hexa β(3)-peptides was synthesized in order to determine the influence of sequence variation as well as staple size and location on conformational stability. From this study we show that appropriately stapled hexa-β(3)-peptides can allow for a number of variations without significant perturbation of the 14-helix.
Organic & Biomolecular Chemistry 02/2012; 10(9):1802-6. · 3.70 Impact Factor
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ABSTRACT: Rtms5 is an deep blue weakly fluorescent GFP-like protein ([Formula: see text], 592 nm; [Formula: see text], 630nm; Φ(F), 0.004) that contains a (66)Gln-Tyr-Gly chromophore tripeptide sequence. We investigated the optical properties and structure of two variants, Rtms5(Y67F) and Rtms5(Y67F/H146S) in which the tyrosine at position 67 was substituted by a phenylalanine. Compared to the parent proteins the optical spectra for these new variants were significantly blue-shifted. Rtms5(Y67F) spectra were characterised by two absorbing species ([Formula: see text], 440 nm and 513 nm) and green fluorescence emission ([Formula: see text], 440 nm; [Formula: see text], 508 nm; Φ(F), 0.11), whilst Rtms5(Y67F/H146S) spectra were characterised by a single absorbing species ([Formula: see text], 440 nm) and a relatively high fluorescence quantum yield (Φ(F,) 0.75; [Formula: see text], 440 nm; [Formula: see text], 508 nm). The fluorescence emissions of each variant were remarkably stable over a wide range of pH (3-11). These are the first GFP-like proteins with green emissions (500-520 nm) that do not have a tyrosine at position 67. The X-ray crystal structure of each protein was determined to 2.2 Å resolution and showed that the benzylidine ring of the chromophore, similar to the 4-hydroxybenzylidine ring of the Rtms5 parent, is non-coplanar and in the trans conformation. The results of chemical quantum calculations together with the structural data suggested that the 513 nm absorbing species in Rtms5(Y67F) results from an unusual form of the chromophore protonated at the acylimine oxygen. These are the first X-ray crystal structures for fluorescent proteins with a functional chromophore containing a phenylalanine at position 67.
PLoS ONE 01/2012; 7(10):e47331. · 4.09 Impact Factor
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ABSTRACT: 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.
Journal of Molecular Recognition 01/2012; 25(1):57-67. · 3.31 Impact Factor
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ABSTRACT: Polycytosine-binding proteins (PCBPs) are triple KH-domain proteins that play an important role in the regulation of translation of eukaryotic mRNA. They are also utilized by viral RNA and have been shown to interact with ssDNA. Underlying their function is the specific recognition of C-rich nucleotides by their KH domains. However, the structural basis of this recognition is only partially understood. Here, the preparation of a His-tagged KH domain is described, representing the first domain of PCBP1 that incorporates a C54S mutation as well as the addition of a C-terminal tryptophan. This construct has facilitated the preparation of highly diffracting crystals in complex with C-rich DNA (sequence ACCCCA). Crystals of the KH1-DNA complex were grown using the hanging-drop vapour-diffusion method in 0.1 M phosphate-citrate pH 4.2, 40%(v/v) PEG 300. X-ray diffraction data were collected to 1.77 Å resolution and the diffraction was consistent with space group P2(1), with unit-cell parameters a = 38.59, b = 111.88, c = 43.42 Å, α = γ = 90.0, β = 93.37°. The structure of the KH1-DNA complex will further our insight into the basis of cytosine specificity by PCBPs.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2011; 67(Pt 10):1257-61. · 0.51 Impact Factor
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ABSTRACT: Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluorescent protein (GFP) that exhibits useful negative reversible photoswitching properties. eCGP123 was derived by the application of both a consensus engineering approach and a recursive evolutionary process. Diffraction-quality crystals of recombinant eCGP123 were obtained by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The eCGP123 crystal diffracted X-rays to 2.10 Å resolution. The data were indexed in space group P1, with unit-cell parameters a = 74.63, b = 75.38, c = 84.51 Å, α = 90.96, β = 89.92, γ = 104.03°. The Matthews coefficient (V(M) = 2.26 Å(3) Da(-1)) and a solvent content of 46% indicated that the asymmetric unit contained eight eCGP123 molecules.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2011; 67(Pt 10):1266-8. · 0.51 Impact Factor
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Nigus D Ambaye,
Stephanie C Pero,
Menachem J Gunzburg,
MinYin Yap,
Daniel J Clayton,
Mark P Del Borgo,
Patrick Perlmutter,
Marie-Isabel Aguilar,
Girja S Shukla,
Elena Peletskaya,
Michelle M Cookson,
David N Krag, Matthew C J Wilce,
Jacqueline A Wilce
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ABSTRACT: Growth-receptor-bound protein (Grb)7 is an adapter protein aberrantly overexpressed, along with the erbB-2 receptor in breast cancer and in other cancers. Normally recruited to focal adhesions with a role in cell migration, it is associated with erbB-2 in cancer cells and is found to exacerbate cancer progression via stimulation of cell migration and proliferation. The G7-18NATE peptide (sequence: WFEGYDNTFPC cyclized via a thioether bond) is a nonphosphorylated peptide that was developed for the specific inhibition of Grb7 by blocking its SH2 domain. Cell-permeable versions of G7-18NATE are effective in the reduction of migration and proliferation in Grb7-overexpressing cells. It thus represents a promising starting point for the development of a therapeutic against Grb7. Here, we report the crystal structure of the G7-18NATE peptide in complex with the Grb7-SH2 domain, revealing the structural basis for its interaction. We also report further rounds of phage display that have identified G7-18NATE analogues with micromolar affinity for Grb7-SH2. These peptides retained amino acids F2, G4, and F9, as well as the YDN motif that the structural biology study showed to be the main residues in contact with the Grb7-SH2 domain. Isothermal titration calorimetry measurements reveal similar and better binding affinity of these peptides compared with G7-18NATE. Together, this study facilitates the optimization of second-generation inhibitors of Grb7.
Journal of Molecular Biology 07/2011; 412(3):397-411. · 4.00 Impact Factor
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ABSTRACT: TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U-rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2'-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways.
Nucleic Acids Research 02/2011; 39(3):1117-30. · 8.03 Impact Factor
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Nigus D Ambaye,
Reece C C Lim,
Daniel J Clayton,
Menachem J Gunzburg,
John T Price,
Stephanie C Pero,
David N Krag, Matthew C J Wilce,
Marie-Isabel Aguilar,
Patrick Perlmutter,
Jacqueline A Wilce
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ABSTRACT: Grb7 is an adapter protein found to be overexpressed in several breast and other cancer cell types along with ErbB2. Grb7 is normally an interaction partner with focal adhesion kinase and in cancer cells also aberrantly interacts with ErbB2. It is thus implicated in the migratory and proliferative potential of cancer cells. Previous studies have shown that the phage display-derived cyclic nonphosphorylated inhibitor peptide, G7-18NATE, when linked to Penetratin, is able to interfere with the interaction of Grb7 with its upstream binding partners and to impact on both cell migration and proliferation. Here we report the synthesis of a biotinylated G7-18NATE covalently attached to just the last seven residues of Penetratin (G7-18NATE-P-Biotin). We demonstrate that this construct is taken up efficiently into MDA-MB-468 breast cancer cells and colocalizes with Grb7 in the cytoplasm. We also used isothermal titration calorimetry to determine the binding affinity of G7-18NATE-P-Biotin to the Grb7-SH2 domain, and showed that it binds with micromolar affinity (K(d) = 14.4 microM), similar to the affinity of G7-18NATE (K(d) = 35.4 microM). Together this shows that this shorter G7-18NATE-P-Biotin construct is suitable for further studies of the antiproliferative and antimigratory potential of this inhibitor.
Biopolymers 01/2011; 96(2):181-8. · 2.87 Impact Factor
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ABSTRACT: Growth factor receptor bound protein 7 (Grb7) is an adapter protein that functions as a downstream effector of growth factor mediated signal transduction. Over-expression of Grb7 has been implicated in a variety of cancers such as breast, blood, pancreatic, esophageal, and gastric carcinomas. Inhibition of Grb7 has been shown to reduce the migratory and proliferative potential of these cancers, making it an attractive therapeutic target. Starting with a known peptide antagonist, the present work reports the application of a succession of computational ligand design tools comprising a ligand shape based similarity search, molecular docking and a 2D-similarity search to identify small molecular antagonists of the Grb7-SH2 domain from the NCI chemical database. Binding to the Grb7-SH2 domain was then experimentally tested using melting point shift assays and isothermal titration calorimetry. Overall, a total of 11 benzopyrazine based small molecular antagonists were identified with affinity for the Grb7-SH2 domain. Representative compounds tested using ITC were revealed to possess moderate binding affinity in the low micromolar range. Finally, the lead compound (NSC642056) was found to reduce the growth of a Grb7-expressing breast cancer cell line with an IC(50) of 86μM. It is expected that the identified antagonists will be useful additions to further explore the function of Grb7 and for the development of inhibitors with therapeutic potential.
Bioorganic & medicinal chemistry 01/2011; 19(1):693-701. · 2.82 Impact Factor
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Nigus D. Ambaye,
Reece C. C. Lim,
Daniel J. Clayton,
Menachem J. Gunzburg,
John T. Price,
Stephanie C. Pero,
David N. Krag, Matthew C. J. Wilce,
Marie-Isabel Aguilar,
Patrick Perlmutter,
Jacqueline A. Wilce
[show abstract]
[hide abstract]
ABSTRACT: Grb7 is an adapter protein found to be overexpressed in several breast and other cancer cell types along with ErbB2. Grb7 is normally an interaction partner with focal adhesion kinase and in cancer cells also aberrantly interacts with ErbB2. It is thus implicated in the migratory and proliferative potential of cancer cells. Previous studies have shown that the phage display-derived cyclic nonphosphorylated inhibitor peptide, G7-18NATE, when linked to Penetratin©, is able to interfere with the interaction of Grb7 with its upstream binding partners and to impact on both cell migration and proliferation. Here we report the synthesis of a biotinylated G7-18NATE covalently attached to just the last seven residues of Penetratin© (G7-18NATE-P-Biotin). We demonstrate that this construct is taken up efficiently into MDA-MB-468 breast cancer cells and colocalizes with Grb7 in the cytoplasm. We also used isothermal titration calorimetry to determine the binding affinity of G7-18NATE-P-Biotin to the Grb7-SH2 domain, and showed that it binds with micromolar affinity (Kd = 14.4 μM), similar to the affinity of G7-18NATE (Kd = 35.4 μM). Together this shows that this shorter G7-18NATE-P-Biotin construct is suitable for further studies of the antiproliferative and antimigratory potential of this inhibitor. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 96: 181–188, 2011.
Biopolymers 12/2010; 96(2):181 - 188. · 2.87 Impact Factor
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ABSTRACT: Grb7 is an adapter protein that is involved in signalling pathways that mediate eukaryotic cell proliferation and migration. Its overexpression in several cancer types has implicated it in cancer progression and led to the development of the G7-18NATE cyclic peptide inhibitor. Here, the preparation of crystals of G7-18NATE in complex with its Grb7 SH2 domain target is reported. Crystals of the complex were grown by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant at room temperature. X-ray diffraction data were collected from crystals to 2.4 Å resolution using synchrotron X-ray radiation at 100 K. The diffraction was consistent with space group P2(1), with unit-cell parameters a=52.7, b=79.1, c=54.7 Å, α=γ=90.0, β=104.4°. The structure of the G7-18NATE peptide in complex with its target will facilitate the rational development of Grb7-targeted cancer therapeutics.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 12/2010; 66(Pt 12):1640-3. · 0.51 Impact Factor
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Siew Siew Pang,
Richard Berry,
Zhenjun Chen,
Lars Kjer-Nielsen,
Matthew A Perugini,
Glenn F King,
Christina Wang,
Sock Hui Chew,
Nicole L La Gruta,
Neal K Williams,
Travis Beddoe,
Tony Tiganis,
Nathan P Cowieson,
Dale I Godfrey,
Anthony W Purcell, Matthew C J Wilce,
James McCluskey,
Jamie Rossjohn
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ABSTRACT: The pre-T-cell antigen receptor (pre-TCR), expressed by immature thymocytes, has a pivotal role in early T-cell development, including TCR β-selection, survival and proliferation of CD4(-)CD8(-) double-negative thymocytes, and subsequent αβ T-cell lineage differentiation. Whereas αβTCR ligation by the peptide-loaded major histocompatibility complex initiates T-cell signalling, pre-TCR-induced signalling occurs by means of a ligand-independent dimerization event. The pre-TCR comprises an invariant α-chain (pre-Tα) that pairs with any TCR β-chain (TCRβ) following successful TCR β-gene rearrangement. Here we provide the basis of pre-Tα-TCRβ assembly and pre-TCR dimerization. The pre-Tα chain comprised a single immunoglobulin-like domain that is structurally distinct from the constant (C) domain of the TCR α-chain; nevertheless, the mode of association between pre-Tα and TCRβ mirrored that mediated by the Cα-Cβ domains of the αβTCR. The pre-TCR had a propensity to dimerize in solution, and the molecular envelope of the pre-TCR dimer correlated well with the observed head-to-tail pre-TCR dimer. This mode of pre-TCR dimerization enabled the pre-Tα domain to interact with the variable (V) β domain through residues that are highly conserved across the Vβ and joining (J) β gene families, thus mimicking the interactions at the core of the αβTCR's Vα-Vβ interface. Disruption of this pre-Tα-Vβ dimer interface abrogated pre-TCR dimerization in solution and impaired pre-TCR expression on the cell surface. Accordingly, we provide a mechanism of pre-TCR self-association that allows the pre-Tα chain to simultaneously 'sample' the correct folding of both the V and C domains of any TCR β-chain, regardless of its ultimate specificity, which represents a critical checkpoint in T-cell development. This unusual dual-chaperone-like sensing function of pre-Tα represents a unique mechanism in nature whereby developmental quality control regulates the expression and signalling of an integral membrane receptor complex.
Nature 10/2010; 467(7317):844-8. · 36.28 Impact Factor
