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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

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The 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 a⁄nity for L-thyroxine and negligible a⁄nity for RBP. The 1.56 A î 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 di¡erences 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. ß

A complex of atypical PKC and Par6 is a common regulator for cell-polarity related processes, which is an essential clue to evolutionary conserved cell-polarity regulation. Here, we determined the crystal structure of the aPKC and Par6 PB1 domain complex to a resolution of 1.5 Å. Both PB1 adopt a ubiquitin fold. aPKC PB1 presents an OPCA motif, 28 amino acid residues with acidic and hydrophobic residues, which interacts with the conserved lysine residue of Par6 PB1 in a front-and-back manner. Structural comparison of the aPKC and Par6 PB1 complex with the p40 phox and p67 phox PB1 complex, subunits of neutrophil NADPH oxidase, reveals that the specific interaction is achieved by tilting the interface so that the insertion or extension in the sequence is engaged in the specificity determinant. The PB1 domain develops the interaction surface on the ubiquitin fold to increase the versatility of molecular interaction.

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