Distinctive binding and structural properties of piscine transthyretin

AbstractThe thyroid hormone binding protein transthyretin (TTR) forms a macromolecular complex with the retinol-specific carrier retinol binding protein (RBP) in the blood of higher vertebrates. Piscine TTR is shown here to exhibit high binding affinity for L-thyroxine and negligible affinity for RBP. The 1.56 Å resolution X-ray structure of sea bream TTR, compared with that of human TTR, reveals a high degree of conservation of the thyroid hormone binding sites. In contrast, some amino acid differences in discrete regions of sea bream TTR appear to be responsible for the lack of protein–protein recognition, providing evidence for the crucial role played by a limited number of residues in the interaction between RBP and TTR. Overall, this study makes it possible to draw conclusions on evolutionary relationships for RBPs and TTRs of phylogenetically distant vertebrates

The impact of binaries on the evolution of star clusters from turbulent molecular clouds

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Crystal structure of peach Pru p 3, the prototypic member of the family of plant non-specific lipid transfer protein pan-allergens

This study describes the three-dimensional crystal structure of a nonspecific lipid transport protein (ns-LTP) from Rosaceae. Whilst ns-LTPs from species other than Rosaceae, such as nuts, cereals, grape, oranges and vegetables are also responsible for plant food allergies, this is less frequent compared with ns-LTPs from Rosaceae in the Mediterranean area. In this heterologously expressed peach Pru p3, a ligand is present inside the central cavity of the protein, presumably a fatty acid that was present or produced in the culture medium of the expression organism Escherichia coli. Moreover, the two molecules of ns-LTP present in the asymmetric unit bind this ligand in a different way, suggesting a significant degree of plasticity for the peach ns-LTP binding cavity despite the presence of four disulphide bridges. Two molecules are present in the asymmetric unit: molecule A is a fully liganded protein, while molecule B apparently represents a partially liganded state. Also, molecular dynamics simulation, along with other evidence, suggests that these two molecular conformations represent different states in solution. Comparison of the 3D models of different ns-LTPs justifies the evidence of a high degree of conservation of the putative IgE binding epitopes among proteins of the Rosaceae family and the presence of significant amino acid replacements in correspondence of the same regions in ns-LTPs of botanical species unrelated to Rosaceae

Structural and mutational analyses of protein-protein interactions between transthyretin and retinol-binding protein

Transthyretin is a tetrameric binding protein involved in the transport of thyroid hormones and in the cotransport of retinol by forming a complex in plasma with retinol-binding protein. in the present study, we report the crystal structure of a macromolecular complex, in which human transthyretin, human holo-retinol-binding protein and a murine anti-retinol-binding protein Fab are assembled according to a 1 : 2 : 2 stoichiometry. the main interactions, both polar and apolar, between retinol-binding protein and transthyretin involve the retinol hydroxyl group and a limited number of solvent exposed residues. the relevance of transthyretin residues in complex formation with retinol-binding protein has been examined by mutational analysis, and the structural consequences of some transthyretin point mutations affecting protein-protein recognition have been investigated. Despite a few exceptions, in general, the substitution of a hydrophilic for a hydrophobic side chain in contact regions results in a decrease or even a loss of binding affinity, thus revealing the importance of interfacial hydrophobic interactions and a high degree of complementarity between retinol-binding protein and transthyretin. the effect is particularly evident when the mutation affects an interacting residue present in two distinct subunits of transthyretin participating simultaneously in two interactions with a retinol-binding protein molecule. This is the case of the amyloidogenic I84S replacement, which abolishes the interaction with retinol-binding protein and is associated with an altered retinol-binding protein plasma transport in carriers of this mutation. Remarkably, some of the residues in mutated human transthyretin that weaken or abolish the interaction with retinol-binding protein are present in piscine transthyretin, consistent with the lack of interaction between retinol-binding protein and transthyretin in fish.PRIN Projects of the 'Ministero dell'Universita e della Ricerca' (Rome, Italy)Universities of Parma and Padua, ItalyUniv Parma, Dept Biochem & Mol Biol, I-43100 Parma, ItalyUniv Padua, Inst Biomol Chem, CNR, I-35131 Padua, ItalyUniv Padua, Dept Chem Sci, I-35131 Padua, ItalyVenetian Inst Mol Med, Padua, ItalyUniversidade Federal de São Paulo, Dept Med, São Paulo, BrazilUniv Padua, Dept Expt Vet Sci, I-35100 Padua, ItalyUniv Padua, CRIBI, I-35100 Padua, ItalyUniversidade Federal de São Paulo, Dept Med, São Paulo, BrazilWeb of Scienc

Expression, purification, and inhibition of human RET tyrosine kinase

Tyrosine kinases are emerging as frequent targets of primary oncogenic events and therefore represent an optimal focus of therapeutical intervention. Genetic alterations that cause dysregulated activation of the RET tyrosine kinase are responsible for a significant fraction of thyroid carcinomas. In an effort towards therapeutic RET inactivation, we have developed a method for expression and purification of recombinant RET catalytic domain for structural purposes and for use in the screening of potential inhibitors of RET kinase activity. His-tagged RET kinase domain was purified from Sf9 insect cell lysate using a two-step chromatographic protocol and characterised. Purified recombinant RET phosphorylated itself and exogenous substrates at physiological pH. A specific peptide substrate, derived from RET activation loop, was identified and experimentally validated. These reagents were used to develop a rapid ELISA-based kinase assay for screening potential inhibitors. Novel RET inhibitors were identified using this assay

Solvent polarity controls the helical conformation of short peptides rich in Calpha-tetrasubstituted amino acids

The two peptides, rich in Calfatetrasubstituted amino acids, Ac-[AibL-(alfaMe)Val-Aib]2-l-His-NH2 (1) and Ac-[Aib-L-(alfaMe)Val-Aib]2-O-tBu (2a) are prevalently helical. They present the unique property of changing their conformation from the alfa- to the 3 10-helix as a function of the polarity of the solvent: a in more polar solvents, 3 10 in less polar ones. Conclusive evidence of this reversible change of conformation is reported on the basis of the circular dichroism (CD) spectra and a detailed two-dimensional NMR analysis in two solvents (trifluoroethanol and methanol) refined with molecular dynamics calculations. The X-ray diffractometric analysis of the crystals of both peptides reveals that they assume a prevalent 3 10-helix conformation in the solid state. This conformation is practically superimposable on that obtained from the NMR analysis of 1 in methanol. The NMR results further validate the reported CD signature of the 3 10-helix and the use of the CD technique for its assessment