The Crystal Structure of the Transthyretin-like Protein from Salmonella dublin, a Prokaryote 5-Hydroxyisourate Hydrolase (2GPZ)

The mechanism of binding of thyroid hormones by the transport protein transthyretin (TTR) in vertebrates is structurally well characterised. However, a homologous family of transthyretin-like proteins (TLPs) present in bacteria as well as eukaryotes do not bind thyroid hormones, instead they are postulated to perform a role in the purine degradation pathway and function as 5-hydroxyisourate hydrolases. Here we describe the 2.5 Angstroms X-ray crystal structure of the TLP from the Gram-negative bacterium Salmonella dublin, and compare and contrast its structure with vertebrate TTRs. The overall architecture of the homotetramer is conserved and, despite low sequence homology with vertebrate TTRs, structural differences within the monomer are restricted to flexible loop regions. However, sequence variation at the dimer-dimer interface has profound consequences for the ligand binding site and provides a structural rationalisation for the absence of thyroid hormone binding affinity in bacterial TLPs: the deep, negatively charged thyroxine-binding pocket that characterises vertebrate TTR contrasts with a shallow and elongated, positively charged cleft in S. dublin TLP. We have demonstrated that Sdu_TLP is a 5-hydroxyisourate hydrolase. Furthermore, using site-directed mutagenesis, we have identified three conserved residues located in this cleft that are critical to the enzyme activity. Together our data reveal that the active site of Sdu_TLP corresponds to the thyroxine binding site in TTRs.<br>

脊椎动物体内转运蛋白运甲状腺素蛋白（transthyretin, TTR）结合甲状腺激素的机制已在结构层面得到充分表征。然而，在细菌及真核生物中存在的同源运甲状腺素蛋白样蛋白（transthyretin-like proteins, TLPs）家族并不结合甲状腺激素，目前推测其在嘌呤降解通路中发挥功能，作为5-羟基异尿酸水解酶行使催化作用。本文报道了来自革兰氏阴性菌都柏林沙门氏菌（Salmonella dublin）的TLP的2.5埃X射线晶体结构，并将其结构与脊椎动物TTR进行对比分析。同源四聚体的整体架构保守，尽管与脊椎动物TTR的序列同源性较低，但单体内部的结构差异仅局限于柔性环区域。然而，二聚体-二聚体界面处的序列变异对配体结合位点具有显著影响，从结构层面合理解释了细菌TLP不具备甲状腺激素结合亲和力的原因：脊椎动物TTR标志性的深凹陷负电荷甲状腺素结合口袋，与都柏林沙门氏菌TLP中浅而细长的正电荷裂隙形成鲜明对比。我们已证实Sdu_TLP为5-羟基异尿酸水解酶。此外，通过定点诱变技术，我们鉴定出该裂隙中三个保守残基，其对酶活性至关重要。综上，本研究结果表明，Sdu_TLP的活性位点对应于TTR中的甲状腺素结合位点。