Phosphorylation of threonine residues on Shc promotes ligand binding and mediates crosstalk between MAPK and Akt pathways in breast cancer cells.

Scaffold proteins play important roles in regulating signalling network fidelity, the absence of which is often the basis for diseases such as cancer. In the present work, we show that the prototypical scaffold protein Shc is phosphorylated by the extracellular signal-regulated kinase, Erk. In addition, Shc threonine phosphorylation is specifically up-regulated in two selected triple-negative breast cancer (TNBC) cell lines. To explore how Erk-mediated threonine phosphorylation on Shc might play a role in the dysregulation of signalling events, we investigated how Shc affects pathways downstream of EGF receptor. Using an in vitro model and biophysical analysis, we show that Shc threonine phosphorylation is responsible for elevated Akt and Erk signalling, potentially through the recruitment of the 14-3-3 ζ and Pin-1 proteins.

Grb2 depletion under non-stimulated conditions inhibits PTEN, promotes Akt-induced tumor formation and contributes to poor prognosis in ovarian cancer.

In the absence of extracellular stimulation the adaptor protein growth factor receptor-bound protein (Grb2) and the phospholipase Plcγ1 compete for the same binding site on fibroblast growth factor receptor 2 (FGFR2). Reducing cellular Grb2 results in upregulation of Plcγ1 and depletion of the phospholipid PI(4,5)P2. The functional consequences of this event on signaling pathways are unknown. We show that the decrease in PI(4,5)P2 level under non-stimulated conditions inhibits PTEN activity leading to the aberrant activation of the oncoprotein Akt. This results in excessive cell proliferation and tumor progression in a xenograft mouse model. As well as defining a novel mechanism of Akt phosphorylation with important therapeutic consequences, we also demonstrate that differential expression levels of FGFR2, Plcγ1 and Grb2 correlate with patient survival. Oncogenesis through fluctuation in the expression levels of these proteins negates extracellular stimulation or mutation and defines them as novel prognostic markers in ovarian cancer.

Making ends meet: the importance of the N- and C-termini for the structure, stability, and function of the third SH3 domain of CIN85.

SH3 domains are common structure, interaction, and regulation modules found in more than 200 human proteins. In this report, we studied the third SH3 domain from the human CIN85 adaptor protein, which plays an important role in both receptor tyrosine kinase downregulation and phosphatidylinositol 3 kinase inhibition. The structure of this domain includes an additional 90° kink after the last canonical ß-strand and features unusual interactions between the termini well outside the boundaries of the standard SH3 domain definition. The extended portions of the domain are well-structured and held together entirely by side chain-side chain interactions. Extensive expression screening showed that these additional contacts provide significantly increased stability to the domain. A similar 90° kink is found in only one other SH3 domain structure, while side chain contacts linking the termini have never been described before. As a result of the increased size of CIN85 SH3 domain C, the proximal proline rich region is positioned such that a possible intramolecular interaction is structurally inhibited. Using the key interactions of the termini as the basis for sequence analysis allowed the identification of several SH3 domains with flanking sequences that could adopt similar structures. This work illustrates the importance of careful experimental analysis of domain boundaries even for a well-characterized fold such as the SH3 domain.

A complex of Shc and Ran-GTPase localises to the cell nucleus.

The three isoforms of the adaptor protein Shc play diverse roles in cell signalling. For example, the observation of p46 Shc in the nuclei of hepatocellular carcinoma cells suggests a function quite distinct from the better characterised cytoplasmic role. Ligands responsible for the transport of various Shc isoforms into organelles such as the nucleus have yet to be reported. To identify such ligands a far western approach was used to determine the p52 Shc interactome. The Ran-GTPase nuclear transport protein was identified and found to bind to p52 Shc in vitro with low micromolar affinity. Co-immunoprecipitation, pull down and fluorescence lifetime imaging microscopy experiments in stable cells confirmed cellular interaction and nuclear localisation. The nuclear transport factor protein NTF2, which functions in cohort with Ran, was shown to form a complex with both RAN and Shc, suggesting a mechanism for Shc entry into the nucleus as part of a tertiary complex.

StpA protein from Escherichia coli condenses supercoiled DNA in preference to linear DNA and protects it from digestion by DNase I and EcoKI.

The nucleoid-associated protein, StpA, of Escherichia coli binds non-specifically to double-stranded DNA (dsDNA) and apparently forms bridges between adjacent segments of the DNA. Such a coating of protein on the DNA would be expected to hinder the action of nucleases. We demonstrate that StpA binding hinders dsDNA cleavage by both the non-specific endonuclease, DNase I, and by the site-specific type I restriction endonuclease, EcoKI. It requires approximately one StpA molecule per 250-300 bp of supercoiled DNA and approximately one StpA molecule per 60-100 bp on linear DNA for strong inhibition of the nucleases. These results support the role of StpA as a nucleoid-structuring protein which binds DNA segments together. The inhibition of EcoKI, which cleaves DNA at a site remote from its initial target sequence after extensive DNA translocation driven by ATP hydrolysis, suggests that these enzymes would be unable to function on chromosomal DNA even during times of DNA damage when potentially lethal, unmodified target sites occur on the chromosome. This supports a role for nucleoid-associated proteins in restriction alleviation during times of cell stress.

Halophilic adaptation of protein-DNA interactions.

Pyrococcus woesei ( Pw ) is an archaeal organism adapted to living in conditions of elevated salt and temperature. Thermodynamic data reveal that the interaction between the TATA-box-binding protein (TBP) from this organism and DNA has an entirely different character to the same interaction in mesophilic counterparts. In the case of the Pw TBP, the affinity of its interaction with DNA increases with increasing salt concentration. The opposite effect is observed in all known mesophilic protein-DNA interactions. The halophilic behaviour can be attributed to sequestration of cations into the protein-DNA complex. By mutating residues in the Pw TBP DNA-binding site, potential sites of cation interaction can be removed. These mutations have a significant effect on the binding characteristics, and the halophilic nature of the Pw TBP-DNA interaction can be reversed, and made to resemble that of a mesophile, in just three mutations. The genes of functionally homologous proteins in organisms existing in different environments show that adaptation is most often accompanied by mutation of an existing protein. However, the importance of any individual residue to a phenotypic characteristic is usually difficult to assess amongst the multitude of changes that occur over evolutionary time. Since the halophilic nature of this protein can be attributed to only three mutations, this reveals that the important phenotype of halophilicity could be rapidly acquired in evolutionary time.

Specific DNA recognition by the type II restriction endonuclease MunI: the effect of pH.

To investigate the effect of pH on sequence-specific binding, a thermodynamic characterization of the interaction of the protein MunI with a specific, and a nonspecific, oligonucleotide was performed. MunI is a type II restriction endonuclease which is able to bind specifically, but loses its enzymatic activity in the absence of magnesium ions. Comparison of the specific and nonspecific interactions at 10 and 25 degrees C shows that the latter is accompanied by a small change in enthalpy, and a negligible change in constant pressure heat capacity. On going through the pH range 5.75-9.0 at 25 degrees C, the affinity of specific complex formation is reduced by 20-fold. The interaction is accompanied by the protonation of groups assumed to be on the protein. Based on the simplest model that will fit the data, two distinct protonation events are observed. At low pH, two groups per protein molecule undergo protonation with a pK(a) of 6.0 and 6.9 in the free and bound forms, respectively. At high pH, a further independent protonation occurs involving two groups with pK(a) values of 8.9 and approximately 10.7 in the free and bound forms, respectively. The change in heat capacity ranges from -2.7 to -1.7 kJ mol(-1) K(-1) in going from pH 6.5 to 8.5. This range of variation of change in heat capacity can be accounted for by the effects of protonation of the interacting molecules. The change in heat capacity, calculated from surface area burial using a previously established relationship (1.15 kJ mol(-1) K(-1)), does not correlate well with the experimentally determined values.

Inhibitors to the Src SH2 domain: a lesson in structure--thermodynamic correlation in drug design.

Src homology 2 (SH2) domains play a key role in many tyrosine kinase-mediated intracellular signal transduction pathways. Aberrancies in the interaction of these domains can lead to a range of disease states. As a result, the pharmaceutical industry has made a large temporal and financial investment in the development of specific inhibitors to these domains. Focusing on the interactions of the SH2 domain from the protein Src, we report how the correlation of structural and thermodynamic data allows an assessment of the process of drug design. The binding site of the protein includes two pockets; one interacts with phosphotyrosine groups on cognate ligands, and the other accommodates an aliphatic hydrophobic side chain. The interaction with cognate ligands is also mediated by a network of water molecules. Thermodynamic data from isothermal titration calorimetric studies suggest that modification of the interactions in the SH2 binding site has been largely unsuccessful in producing high-affinity inhibitors. Furthermore, it appears that compounds that disrupt the interfacial water pay the price for the loss of the contribution to the free energy from a network of hydrogen bonds.

Site-specific cation binding mediates TATA binding protein-DNA interaction from a hyperthermophilic archaeon.

Pyrococcus woesei (Pw) is a hyperthermophilic archaeal organism that exists under conditions of high salt and elevated temperature. In a previous study [O'Brien, R., DeDecker, B., Fleming, K., Sigler, P. B., and Ladbury, J. E., (1998) J. Mol. Biol. 279, 117-125], we showed that, despite the similarity of primary and secondary structure, the TATA box binding protein (TBP) from Pw binds thermodynamically in a fundamentally different way to its mesophilic counterparts. The affinity of the interaction increases as the salt concentration is increased. The formation of the protein-DNA complex involves the release of water and the uptake of ions, which were hypothesized to be cations. Here we test this hypothesis by selecting potential cation binding sites at negatively charged, acidic residues in the complex interface. These were substituted using site-directed mutagenesis of specific residues. Changes in the thermodynamic parameters on formation of the mutant protein-DNA complex were determined using isothermal titration calorimetry and compared to the wild type interaction. Removal of a glutamate residue from the binding site resulted in the uptake of one less cation on formation of the complex. This glutamate (E12) is directly involved in the binding of cations in the complex interface. Substitution of another acidic residue proximal to the DNA binding site (D101) had no effect on cation uptake, suggesting that the location of the amino acid on the protein surface is important in dictating the potential to coordinate cations. Removal of the cation binding site provided a more favorable entropy of binding; however, this effect is significantly reduced at higher salt concentrations. The removal of the cation binding site led to an increase in affinity with respect to the wild-type TBP at low salt concentrations.

A structural characterization of the interactions between titin Z-repeats and the alpha-actinin C-terminal domain.

Titin and alpha-actinin, two modular muscle proteins, are with actin the major components of the Z-band in vertebrate striated muscles where they serve to organize the antiparallel actin filament arrays in adjacent sarcomeres and to transmit tension between sarcomeres during activation. Interactions between titin and alpha-actinin have been mainly localized in a 45-amino acid multiple motif (Z-repeat) in the N-terminal region of titin and the C-terminal region of alpha-actinin. In this study, we provide the first quantitative characterization of alpha-actinin-Z-repeat recognition and dissect the interaction to its minimal units. Different complementary techniques, such as circular dichroism, calorimetry, and nuclear magnetic spectroscopy, were used. Two overlapping alpha-actinin constructs (Act-EF34 and Act-EF1234) containing two and four EF-hand motifs, respectively, were produced, and their folding properties were examined. Complex formation of Act-EF34 and Act-EF1234 with single- and double-Z-repeat constructs was studied. Act-EF34 was shown quantitatively to be necessary and sufficient for binding to Z-repeats, excluding the presence of additional high-affinity binding sites in the remaining part of the domain. The binding affinities of the different Z-repeats for Act-EF34 range from micromolar to millimolar values. The strongest of these interactions are comparable to those observed in troponin C-troponin I complexes. The binding affinities for Act-EF34 are maximal for Zr1 and Zr7, the two highly homologous sequences present in all muscle isoforms. No cooperative or additional contributions to the interaction were observed for Z-repeat double constructs. These findings have direct relevance for evaluating current models of Z-disk assembly.

The role of the Src homology 3-Src homology 2 interface in the regulation of Src kinases.

The regulatory fragment of Src kinases, comprising Src homology (SH) 3 and SH2 domains, is responsible for controlled repression of kinase activity. We have used a multidisciplinary approach involving crystallography, NMR, and isothermal titration calorimetry to study the regulatory fragment of Fyn (FynSH32) and its interaction with a physiological activator: a fragment of focal adhesion kinase that contains both phosphotyrosine and polyproline motifs. Although flexible, the preferred disposition of SH3 and SH2 domains in FynSH32 resembles the inactive forms of Hck and Src, differing significantly from LckSH32. This difference, which results from variation in the SH3-SH2 linker sequences, will affect the potential of the regulatory fragments to repress kinase activity. This surprising result implies that the mechanism of repression of Src family members may vary, explaining functional distinctions between Fyn and Lck. The interaction between FynSH32 and focal adhesion kinase is restricted to the canonical SH3 and SH2 binding sites and does not affect the dynamic independence of the two domains. Consequently, the interaction shows no enhancement by an avidity effect. Such an interaction may have evolved to gain specificity through an extended recognition site while maintaining rapid dissociation after signaling.

Structural characterization of the N-terminal oligomerization domain of the bacterial chromatin-structuring protein, H-NS.

The H-NS protein plays a key role in condensing DNA and modulating gene expression in bacterial nucleoids. The mechanism by which this is achieved is dependent, at least in part, on the oligomerization of the protein. H-NS consists of two distinct domains; the N-terminal domain responsible for protein oligomerization, and the C-terminal DNA binding domain, which are separated by a flexible linker region. We present a multidimensional NMR study of the amino-terminal 64 residues of H-NS (denoted H-NS1-64) from Salmonella typhimurium, which constitute the oligomerization domain. This domain exists as a homotrimer, which is predicted to be self-associated through a coiled-coil configuration. NMR spectra show an equivalent magnetic environment for each monomer indicating that the polypeptide chains are arranged in parallel with complete 3-fold symmetry. Despite the limited resonance dispersion, an almost complete backbone assignment for 1H(N), 1H(alpha), 15N, 13CO and 13C(alpha) NMR resonances was obtained using a suite of triple resonance experiments applied to uniformly 15N-, 13C/15N- and 2H/13C/15N-labelled H-NS1-64 samples. The secondary structure of H-NS1-64 has been identified on the basis of the analysis of 1H(alpha), 13C(alpha), 13Cbeta and 13CO chemical shifts, NH/solvent exchange rates, intra-chain H(N)-H(N) and medium-range nuclear Overhauser enhancements (NOEs). Within the context of the homotrimer, each H-NS1-64 protomer consists of three alpha-helices spanning residues 2-8, 12-20 and 22-53, respectively. A topological model is presented for the symmetric H-NS1-64 trimer based upon the combined analysis of the helical elements and the pattern of backbone amide group 15N nuclear relaxation rates within the context of axially asymmetric diffusion tensor. In this model, the longest of the three helices (helix 3, residues 22-53) forms a coiled-coil interface with the other chains in the homotrimer. The two shorter N-terminal helices fold back onto the outer surface of the coiled-coil core and potentially act to stabilise this configuration.

Comparison of binding energies of SrcSH2-phosphotyrosyl peptides with structure-based prediction using surface area based empirical parameterization.

The prediction of binding energies from the three-dimensional (3D) structure of a protein-ligand complex is an important goal of biophysics and structural biology. Here, we critically assess the use of empirical, solvent-accessible surface area-based calculations for the prediction of the binding of Src-SH2 domain with a series of tyrosyl phosphopeptides based on the high-affinity ligand from the hamster middle T antigen (hmT), where the residue in the pY+ 3 position has been changed. Two other peptides based on the C-terminal regulatory site of the Src protein and the platelet-derived growth factor receptor (PDGFR) are also investigated. Here, we take into account the effects of proton linkage on binding, and test five different surface area-based models that include different treatments for the contributions to conformational change and protein solvation. These differences relate to the treatment of conformational flexibility in the peptide ligand and the inclusion of proximal ordered solvent molecules in the surface area calculations. This allowed the calculation of a range of thermodynamic state functions (deltaCp, deltaS, deltaH, and deltaG) directly from structure. Comparison with the experimentally derived data shows little agreement for the interaction of SrcSH2 domain and the range of tyrosyl phosphopeptides. Furthermore, the adoption of the different models to treat conformational change and solvation has a dramatic effect on the calculated thermodynamic functions, making the predicted binding energies highly model dependent. While empirical, solvent-accessible surface area based calculations are becoming widely adopted to interpret thermodynamic data, this study highlights potential problems with application and interpretation of this type of approach. There is undoubtedly some agreement between predicted and experimentally determined thermodynamic parameters: however, the tolerance of this approach is not sufficient to make it ubiquitously applicable.

The ATPase cycle of Hsp90 drives a molecular 'clamp' via transient dimerization of the N-terminal domains.

How the ATPase activity of Heat shock protein 90 (Hsp90) is coupled to client protein activation remains obscure. Using truncation and missense mutants of Hsp90, we analysed the structural implications of its ATPase cycle. C-terminal truncation mutants lacking inherent dimerization displayed reduced ATPase activity, but dimerized in the presence of 5'-adenylamido-diphosphate (AMP-PNP), and AMP-PNP- promoted association of N-termini in intact Hsp90 dimers was demonstrated. Recruitment of p23/Sba1 to C-terminal truncation mutants also required AMP-PNP-dependent dimerization. The temperature- sensitive (ts) mutant T101I had normal ATP affinity but reduced ATPase activity and AMP-PNP-dependent N-terminal association, whereas the ts mutant T22I displayed enhanced ATPase activity and AMP-PNP-dependent N-terminal dimerization, indicating a close correlation between these properties. The locations of these residues suggest that the conformation of the 'lid' segment (residues 100-121) couples ATP binding to N-terminal association. Consistent with this, a mutation designed to favour 'lid' closure (A107N) substantially enhanced ATPase activity and N-terminal dimerization. These data show that Hsp90 has a molecular 'clamp' mechanism, similar to DNA gyrase and MutL, whose opening and closing by transient N-terminal dimerization are directly coupled to the ATPase cycle.

Localization and characterization of the hyaluronan-binding site on the link module from human TSG-6.

BACKGROUND: The interactions of hyaluronan (HA) with proteins are important in extracellular matrix integrity and leukocyte migration and are usually mediated by a domain termed a Link module. Although the tertiary structure of a Link module has been determined, the molecular basis of HA-protein interactions remains poorly understood. RESULTS: Isothermal titration calorimetry was used to characterize the interaction of the Link module from human TSG-6 (Link_TSG6) with HA oligosaccharides of defined length (HA(4)-HA(16)). All oligomers bound (except HA(4)) with K(d) values ranging from 0.2-0.5 microM at 25 degrees C. The reaction is exothermic with a favourable entropy and the thermodynamic profile is similar to those of other glycosaminoglycan-protein interactions. The HA(8) recognition site on Link_TSG6 was localized by comparing nuclear magnetic resonance (NMR) spectra from a 1:1 complex with free protein. Residues perturbed on HA binding include both amino acids that are likely to be directly involved in the interaction (i.e., Lys11, Tyr59, Asn67, Phe70, Lys72 and Tyr78) and those affected by a ligand-induced conformational change in the beta4/beta5 loop. The sidechain of Asn67 becomes more rigid in the complex suggesting that it is in close proximity to the binding site. CONCLUSIONS: In TSG-6 a single Link module is sufficient for a high-affinity interaction with HA. The HA-binding surface on Link_TSG6 is found in a similar position to that suggested previously for CD44, indicating that its location might be conserved across the Link module superfamily. Here we find no evidence for the involvement of linear sequence motifs in HA binding.

Characterization of sequence-specific DNA binding by the transcription factor Oct-1.

The DNA-binding domain of the Oct-1 transcription factor, POU, recognizes a defined DNA sequence known as the octamer element to regulate the expression of both general and cell-type-specific genes. The two-part DNA-binding domain partially encircles the DNA to recognize the eight base pairs of the octamer element. We have characterized the binding of Oct-1/POU to an octamer element using isothermal titration calorimetry. As found for other cognate protein/DNA complexes, the formation of the Oct-1 POU/DNA complex is associated with a large negative heat capacity change, DeltaC(p)()(, obs). However, the observed change is much greater than expected by empirical relationships with buried surface area. Supported by data from proteolysis studies on the free and DNA-bound protein, we propose that the discrepancy in heat capacity arises principally from the partial folding of the Oct-1 POU protein upon complex formation. Formation of the Oct-1 POU/DNA complex is strongly dependent on ionic strength, and the detailed quantification of this relationship suggests that six charged contacts are made between the protein and the phosphate groups of the DNA. This agrees with observations from the crystal structure of an Oct-1 POU/DNA complex.

Oligomerization of the chromatin-structuring protein H-NS.

H-NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H-NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H-NS protein from Salmonella typhimurium. The N-terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an alpha-helix, likely to be in coiled-coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full-length protein no single, defined oligomeric state is adopted. The C-terminal 48 residues do not participate in oligomerization and form a monomeric, DNA-binding domain. These N- and C-terminal domains are joined via a flexible linker which enables them to function independently within the context of the full-length protein. This novel mode of oligomerization may account for the unusual binding properties of H-NS.

Alternative modes of binding of proteins with tandem SH2 domains.

The issue of specificity in tyrosine kinase intracellular signaling mediated by src homology 2 (SH2) domains has great importance in the understanding how individual signals maintain their mutual exclusivity and affect downstream responses. Several proteins contain tandem SH2 domains that, on interacting with their ligand, provide a higher level of specificity than can be afforded by the interaction of a single SH2 domain. In this study, we focus on the comparison of two proteins ZAP70 and the p85 subunit of PI 3-kinase, which although distinctly different in function and general structure, possess tandem SH2 domains separated by a linker region and which bind to phosphorylated receptor molecules localized to the cell membrane. Binding studies using isothermal titration calorimetry show that these two proteins interact with peptides mimicking their physiological ligands in very different ways. In the case of the SH2 domains from ZAP70, they interact with a stoichiometry of unity, while p85 is able to make two distinct interactions, one with a stoichiometry of 1:1 and the other with two p85 molecules interacting with one receptor. The observation of two different modes of binding of p85 might be important in providing different cellular responses based on fluctuating intracellular concentration regimes of this protein. Thermodynamic data on both proteins suggest that a conformational change occurs on binding. On investigation of this structural change using a truncated form of p85 (including just the two SH2 domains and the inter-SH2 region), both NMR and circular dichroism spectroscopic studies failed to show significant changes in secondary structure. This suggests that any conformational change associated with binding is small and potentially limited to loop regions of the protein.

Searching for specificity in SH domains.

Elucidating protein-protein interactions has been a central feature to understanding intracellular signal transduction. Many of the binding sites of the interacting proteins in these pathways are within highly sequentially homologous and structurally conserved domains. We challenge the dogma that mutual exclusivity in signalling is derived from a high level of specificity in these domains.

Specificity and interactions of the protein OppA: partitioning solvent binding effects using mass spectrometry.

Mass spectrometry (MS) was used to characterise the binding of the 58 kDa protein OppA to 11 peptides with diverse properties. Peptides with two, three and five amino acid residues were added to OppA, and the mass spectra showed that the highest-affinity complexes are formed between OppA and tripeptide ligands. Lower-affinity complexes were observed for OppA and dipeptide ligands, and no complex formation was detected with pentapeptides or a tripeptide in which the N-terminal amino group was acetylated. Tripeptides containing a single d amino acid residue were found not to bind to native OppA. Evidence from the peak width and the, charge in the spectra of the complexes suggests that the bound peptides are encapsulated by the protein in a solvent-filled cavity in the gas phase of the mass spectrometer. Analysis of the proportions of peptide-bound and free proteins under low-energy MS conditions shows a good correlation with solution-phase K(d) measurements where available. Increasing the internal energy of the gas-phase complex led to dissociation of the complex. The ease of dissociation is interpreted in terms of the intrinsic stability of the complex in the absence of the stabilising effects of bulk solvent. The results from this study demonstrate insensitivity to the hydrophobic and ionic properties, of the side-chains of the peptides, in contrast to the investigation of other protein ligand systems by MS. Moreover, these findings are in accord with the physiological role of this protein in allowing into the cell di- and tripeptides containing naturally occurring amino acids, regardless of their sequence, while barring access to potentially harmful peptide mimics.