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.

脊椎动物体内运甲状腺素蛋白（transthyretin, TTR）结合甲状腺激素的分子机制已在结构层面得到充分表征。然而，广泛存在于细菌与真核生物中的运甲状腺素蛋白样蛋白（transthyretin-like proteins, TLPs）同源家族并不结合甲状腺激素；目前学界推测其参与嘌呤降解通路，功能为5-羟异尿酸水解酶。本研究报道了革兰氏阴性菌都柏林沙门氏菌来源TLP的2.5埃X射线晶体结构，并将其结构与脊椎动物TTR进行了对比分析。同源四聚体的整体折叠模式保守存在，尽管与脊椎动物TTR的序列同源性较低，但单体层面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产生了显著影响，从结构层面合理解释了细菌TLPs无法结合甲状腺激素的原因：脊椎动物TTR标志性的深凹带负电甲状腺素结合口袋，在都柏林沙门氏菌TLP中变为浅而狭长的带正电裂隙。我们已证实Sdu_TLP为5-羟异尿酸水解酶。进一步通过定点突变实验，鉴定出该裂隙内的3个保守残基，其对酶活性至关重要。综上，本研究数据表明Sdu_TLP的活性位点对应TTR中的甲状腺素结合位点。

搜集汇总

背景与挑战

背景概述

该数据集提供了沙门氏菌都柏林株的转甲状腺素样蛋白（TLP）的2.5埃X射线晶体结构，通过对比脊椎动物转甲状腺素（TTR），揭示了其作为5-羟基异尿酸水解酶的功能，并鉴定了三个关键活性位点残基。数据集聚焦于细菌TLP的结构特征和酶活性机制，为理解蛋白质进化与功能分化提供了结构基础。