Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

<p>Abstract</p> <p>Background</p> <p>Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.</p> <p>Results</p> <p>As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K_d= ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.</p> <p>Conclusion</p> <p>Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.</p

Bemestingsproef met stikstof en met kali : resultaten van de derde teelt chrysanten (1973)

<p><b>Copyright information:</b></p><p>Taken from "Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation"</p><p>http://www.biomedcentral.com/1472-6807/7/58</p><p>BMC Structural Biology 2007;7():58-58.</p><p>Published online 25 Sep 2007</p><p>PMCID:PMC2131756.</p><p></p>ture elements present in the Grb7 SH2 structure as determined by WHATIF [71] are shaded from purple at the N-terminus to red at the C-terminus. Secondary structure elements of the canonical SH2 domain as defined by Eck . [41] are shown in green and orange symbols above the sequences. The boundaries of these elements differ slightly from that observed in the Grb7 SH2 domain. Residue number is for the Grb7 SH2 domain (b) Cartoon representation of the Grb7 SH2 domain shaded from purple at the N-terminus to red at the C-terminus. The extended DE loop distinguishes this family of SH2 domains from others. (c) A structural comparison of the Grb7 SH2 domain (green) with the Grb7 SH2 domain bound to an ErbB2 derived phosphopeptide (1MW4; black; [29]). The location of the bound phosphopeptide is indicated

Structural Biology and Its Applications to the Health Sciences

Part of the decipherment of genomic information lies in understanding the structure and function of the protein products of these genes. Protein structure is of further importance because of the molecular basis of many diseases. Structural biology is the field of research focusing on the experimental determination of the structure of biological molecules. We review the field of structural biology and its application to medical research and drug discovery, and describe the structural results recently obtained in our laboratory for the detoxifying enzyme glutathione S-transferase from the Asian mosquito Anopheles dirus species B, an important malaria vector. These enzymes have detoxifying activity toward pesticides and thus contribute to pesticide resistance in insects. Since the first protein structure (of sperm whale myoglobin) was determined (1) and Watson and Crick discovered the double helix structure of DNA (2), there has been an ever-increasing research effort in the field of structural biology. Broadly, structural biology is defined as the investigation of the structure and function of biological systems at the molecular level. The significance of this field of research in part derives from the fact that macromolecular structure is important to many disease states. Sickle-cell anemia was recognized to be a result of a mutation in hemoglobin, causing it to polymerize into long rod-shape complexes that distort and destroy red blood cells (3). The structural consequence of this mutation is now understood (4). Today, many molecular diseases, such as cancer, are known to result from mutations of genes that affect the gene product, altering its function. This is invariably caused by changes in the structure and function of the protein product of the gene. Understanding these processes can be important for treating the disease. Information about protein structure can be used in so-called structure-based drug design, where a macromolecular structure is used as a template for drug design. This review examines the field of structural biology and its relevance to medicine and drug discovery. Typically, protein crystallography and nuclear magnetic resonance (NMR) spectroscopy have been the tools of choice for the structural biologist. Briefly described here are the two main techniques for macromolecular structure determination. X-ray Crystallography X-ray crystallography can give atomic resolution structure of proteins and other macromolecules, such as DNA and their complexes. The technique requires the availability of milligram quantities of &gt;99% pure macromolecule, usually produced through cloning and overexpression in bacterial plasmids, and then purified through the standard techniques of biochemistry, such as gel filtration, affinity chromatography, and ion exchange chromatography. The macromolecule must then be crystalized. This is usually achieved through the addition of the protein to a precipitant, such as ammonium sulfate or polyethylene glycol. Extensive trials of numerous potential conditions are often required to find a condition that gives crystals of sufficiently high quality for X-ray analysis. The next step is to place the crystal in an X-ray beam produced by a laboratory source or at a synchrotron radiation source, such as those located in Trieste (Italy) or Argonne (USA). Crystals diffract X-rays, and the resulting pattern of scattered X-rays is processed computationally to reveal the electron density of the molecule subject. This technique is reviewed in detail elsewhere (5). Nuclear Magnetic Resonance Spectroscopy NMR is another important tool for probing the structure of biological molecules. Like X-ray crystallography, this technique can provide three-dimensional structural information, but the underlying method is completely different. Like X-ray crystallography, the technique requires milligram quantities of www.cmj.hr 37

Structure of the PCBP2/stem-loop IV complex underlying translation initiation mediated by the poliovirus type I IRES.

The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem-loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome

Flexibility revealed by the 1.85 Å crystal structure of the β sliding-clamp subunit of Escherichia coli DNA polymerase III

The subunit of the Escherichia coli replicative DNA polymerase III holoenzyme is the sliding clamp that interacts with the (polymerase) subunit to maintain the high processivity of the enzyme. The protein is a ring-shaped dimer of 40.6 kDa subunits whose structure has previously been determined at a resolution of 2.5 Å [Kong et al. (1992), Cell, 69, 425-437]. Here, the construction of a new plasmid that directs overproduction of to very high levels and a simple procedure for large-scale purification of the protein are described. Crystals grown under slightly modified conditions diffracted to beyond 1.9 Å at 100 K at a synchrotron source. The structure of the dimer solved at 1.85 Å resolution shows some differences from that reported previously. In particular, it was possible at this resolution to identify residues that differed in position between the two subunits in the unit cell; side chains of these and some other residues were found to occupy alternate conformations. This suggests that these residues are likely to be relatively mobile in solution. Some implications of this flexibility for the function of are discussed

Structure of the PCBP2/stem-loop IV complex underlying translation initiation mediated by the poliovirus type I IRES.

The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem-loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome

Quinine Binding by the Cocaine-Binding Aptamer. Thermodynamic and Hydrodynamic Analysis of High-Affinity Binding of an Off-Target Ligand

The cocaine-binding aptamer is unusual in that it tightly binds molecules other than the ligand it was selected for. Here, we study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titration calorimetry was used to quantify the quinine-binding affinity and thermodynamics of a set of sequence variants of the cocaine-binding aptamer. We find that the affinity of the cocaine-binding aptamer for quinine is 30−40 times stronger than it is for cocaine. Competitive binding studies demonstrate that both quinine and cocaine bind at the same site on the aptamer. The ligand-induced structural-switching binding mechanism of an aptamer variant that contains three base pairs in stem 1 is retained with quinine as a ligand. The short stem 1 aptamer is unfolded or loosely folded in the free form and becomes folded when bound to quinine. This folding is confirmed by NMR spectroscopy and by the short stem 1 construct having a more negative change in heat capacity of quinine binding than is seen when stem 1 has six base pairs. Small-angle X-ray scattering (SAXS) studies of the free aptamer and both the quinine- and the cocaine-bound forms show that, for the long stem 1 aptamers, the three forms display similar hydrodynamic properties, and the ab initio shape reconstruction structures are very similar. For the short stem 1 aptamer there is a greater variation among the SAXS-derived ab initio shape reconstruction structures, consistent with the changes expected with its structural-switching binding mechanism

Quinine Binding by the Cocaine-Binding Aptamer. Thermodynamic and Hydrodynamic Analysis of High-Affinity Binding of an Off-Target Ligand

The cocaine-binding aptamer is unusual in that it tightly binds molecules other than the ligand it was selected for. Here, we study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titration calorimetry was used to quantify the quinine-binding affinity and thermodynamics of a set of sequence variants of the cocaine-binding aptamer. We find that the affinity of the cocaine-binding aptamer for quinine is 30−40 times stronger than it is for cocaine. Competitive binding studies demonstrate that both quinine and cocaine bind at the same site on the aptamer. The ligand-induced structural-switching binding mechanism of an aptamer variant that contains three base pairs in stem 1 is retained with quinine as a ligand. The short stem 1 aptamer is unfolded or loosely folded in the free form and becomes folded when bound to quinine. This folding is confirmed by NMR spectroscopy and by the short stem 1 construct having a more negative change in heat capacity of quinine binding than is seen when stem 1 has six base pairs. Small-angle X-ray scattering (SAXS) studies of the free aptamer and both the quinine- and the cocaine-bound forms show that, for the long stem 1 aptamers, the three forms display similar hydrodynamic properties, and the ab initio shape reconstruction structures are very similar. For the short stem 1 aptamer there is a greater variation among the SAXS-derived ab initio shape reconstruction structures, consistent with the changes expected with its structural-switching binding mechanism

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation-1

<p><b>Copyright information:</b></p><p>Taken from "Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation"</p><p>http://www.biomedcentral.com/1472-6807/7/58</p><p>BMC Structural Biology 2007;7():58-58.</p><p>Published online 25 Sep 2007</p><p>PMCID:PMC2131756.</p><p></p>l is coloured blue and negatively charged electrostatic potential is coloured red. The positions of the phosphate binding pocket is indicated. (b) A 2F- Felectron density map depicting the phosphate binding pocket of Grb7 SH2. A sulphate ion co-crystallised in this pocket in all four molecules in the asymmetric unit. The map is contoured at 1 σ. R438, R458, Q461 and S460 form direct contacts with the sulphate ion and are labeled. The side-chain of R462 lacks well defined density and is probably fairly mobile in the crystal