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首页> 外文期刊>Infection and immunity >Intramolecular Interactions between the Protease and Structural Domains Are Important for the Functions of Serine Protease Autotransporters
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Intramolecular Interactions between the Protease and Structural Domains Are Important for the Functions of Serine Protease Autotransporters

机译：蛋白酶和结构域之间的分子内相互作用对于丝氨酸蛋白酶自转运蛋白的功能很重要

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Autotransporter (AT) is a protein secretion pathway found in Gram-negative bacteria featuring a multidomain polypeptide with a signal sequence, a passenger domain, and a translocator domain. An AT subfamily named serine protease ATs of the family Enterobacteriaceae (SPATEs) is characterized by the presence of a conserved serine protease motif in the passenger domain which contributes to bacterial pathogenesis. The goal of the current study is to determine the importance of the passenger domain conserved residues in the SPATE proteolytic and adhesive functions using the temperature-sensitive hemagglutinin (Tsh) protein as our model. To begin, mutations of 21 fully conserved residues in the four passenger domain conserved motifs were constructed by PCR-based site-directed mutagenesis. Seventeen mutants exhibited a wild-type secretion level; among these mutants, eight displayed reduced proteolytic activities in Tsh-specific oligopeptide and mucin cleavage assays. These eight mutants also demonstrated lower affinities to extracellular matrix proteins, collagen IV, and fibronectin. These eight conserved residues were analyzed by molecular graphics modeling to demonstrate their intramolecular interactions with the catalytic triad and other key residues. Additional mutations were made to confirm the above interactions in order to demonstrate their significance to the SPATE functions. Altogether our data suggest that certain conserved residues in the SPATE passenger domain are important for both the proteolytic and adhesive activities of SPATE by maintaining the proper protein structure via intramolecular interactions between the protease and β-helical domains. Here, we provide new insight into the structure-function relationship of the SPATEs and the functional roles of their conserved residues.
机译：自转运蛋白（AT）是在革兰氏阴性细菌中发现的一种蛋白质分泌途径，其特征在于具有信号序列，乘客结构域和易位结构域的多结构域多肽。肠杆菌科（SPATE）的称为丝氨酸蛋白酶AT的AT亚家族的特征在于在客运结构域中存在保守的丝氨酸蛋白酶基序，这有助于细菌发病。当前研究的目标是使用温度敏感血凝素（Tsh）蛋白作为我们的模型，确定SPATE蛋白水解和粘附功能中客体结构域保守残基的重要性。首先，通过基于PCR的定点诱变构建了四个乘客域保守基序中21个完全保守残基的突变。十七个突变体表现出野生型分泌水平。在这些突变体中，有8个在Tsh特异性寡肽和粘蛋白裂解试验中显示出降低的蛋白水解活性。这八个突变体还表现出对细胞外基质蛋白，胶原蛋白IV和纤连蛋白的较低亲和力。通过分子图形建模分析了这八个保守残基，以证明它们与催化三联体和其他关键残基的分子内相互作用。为了证实上述相互作用对SPATE功能的重要性，还进行了其他突变。总的来说，我们的数据表明，SPATE客运结构域中的某些保守残基通过蛋白酶和β-螺旋结构域之间的分子内相互作用维持适当的蛋白质结构，对SPATE的蛋白水解和粘附活性均很重要。在这里，我们提供了对SPATEs的结构-功能关系及其保守残基的功能作用的新见解。

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