Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125
Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125
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DOI：
10.1002/prot.24420
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发表时间：
2014-03-01
影响因子：
2.9
通讯作者：
Banfield, Mark J.
中科院分区：
文献类型：
--
作者：
Walden, Miriam;Crow, Allister;Banfield, Mark J.
化脓性链球菌和其他革兰氏阳性细菌病原体在其表面呈现长的大分子细丝，称为皮利，介导粘附和定植。这些皮利是共价聚合物，由分选酶组装。通常，它们在其尖端包含推定的粘附素、以多个拷贝存在的骨架亚基和共价锚定至细胞表面的肽聚糖层的基底亚基。菌毛蛋白亚基的晶体结构揭示了在这些蛋白质中存在不寻常的共价键，包括分子内异肽和内部硫酯键。骨架菌毛蛋白中的分子内异肽键对蛋白质的稳定性很重要。在这里，使用野生型蛋白和一组突变体，我们评估了S的蛋白水解和热稳定性。在存在和不存在其分子内异肽和内部硫酯键的情况下，我们还确定了内部硫酯键变体Spy 0125 Cys 426 Ala的晶体结构。我们发现，突变的分子内异肽键妥协的Spy 0125的稳定性。使用有限的蛋白水解和热变性试验，我们可以分离每个分子内异肽键对Spy 0125稳定性的贡献。相反，内部硫酯键的突变对蛋白质稳定性的影响较小，晶体结构与野生型基本相同。这项工作表明，Spy 0125中的内部硫酯，虽然有一个小的贡献作用，是不需要蛋白质的稳定性，必须有一个不同的主要功能，最有可能介导与宿主细胞配体的共价相互作用。Proteins 2014; 82：517-527.© 2013 The Authors Proteins：Structure，Function，and Bioinformatics Published by Wiley Periodicals，Inc.
Streptococcus pyogenesand other Gram‐positive bacterial pathogens present long macromolecular filaments known as pili on their surface that mediate adhesion and colonization. These pili are covalent polymers, assembled by sortases. Typically, they comprise a putative adhesin at their tip, a backbone subunit present in multiple copies and a basal subunit that is covalently anchored to the peptidoglycan layer of the cell surface. The crystal structures of pilin subunits revealed the presence of unusual covalent linkages in these proteins, including intramolecular isopeptide and internal thioester bonds. The intramolecular isopeptide bonds in backbone pilins are important for protein stability. Here, using both the wild‐type protein and a set of mutants, we assessed the proteolytic and thermal stability of theS. pyogenespilus tip adhesin Spy0125, in the presence and absence of its intramolecular isopeptide and internal thioester bonds. We also determined a crystal structure of the internal thioester bond variant Spy0125Cys426Ala. We find that mutations in the intramolecular isopeptide bonds compromise the stability of Spy0125. Using limited proteolysis and thermal denaturation assays, we could separate the contribution of each intramolecular isopeptide bond to Spy0125 stability. In contrast, mutation in the internal thioester bond had a lesser effect on protein stability and the crystal structure is essentially identical to wild type. This work suggests that the internal thioester in Spy0125, although having a minor contributory role, is not required for protein stability and must have a different primary function, most likely mediating a covalent interaction with host cell ligands. Proteins 2014; 82:517–527. © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.