Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin
Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin
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DOI：
10.1073/pnas.1316855111
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发表时间：
2014-01-28
影响因子：
11.1
通讯作者：
Baker, Edward N.
中科院分区：
文献类型：
--
作者：
Kwon, Hanna;Squire, Christopher J.;Baker, Edward N.
革兰氏阳性细菌由锚定到细胞壁并从其突出以介导定殖、附着到宿主细胞和发病的各种蛋白质修饰。这些蛋白质和蛋白质组装体，如皮利，通常是长而薄的，但必须承受高水平的机械应力和蛋白水解攻击。最近发现的分子内异肽键交联，形成自催化，在皮利从化脓性链球菌的菌毛突出的作用，这种交联可以发挥稳定这种结构。我们研究了产气荚膜梭菌的一个假定的细胞表面粘附素，它包含一个N-末端粘附素结构域，然后是11个重复结构域。两个结构域片段的晶体结构显示，每个结构域具有IgG样折叠，并含有连接Thr和Gln侧链的前所未有的酯键。MS证实了这些键的存在。我们表明，通过一个相邻的组氨酸残基在丝氨酸蛋白酶样机制催化的自催化分子内反应形成的债券。两个隐藏的酸性残基有助于反应。通过诱变，我们表明，酯键的损失大大降低了热稳定性，增加了对蛋白水解的敏感性。与菌毛蛋白结构域一样，尽管IG折叠类型不同，但这些键位于连接第一条和最后一条链的关键位置。生物信息学分析表明，类似的结构域和酯键交联在革兰氏阳性细菌粘附素中广泛存在。
Gram-positive bacteria are decorated by a variety of proteins that are anchored to the cell wall and project from it to mediate colonization, attachment to host cells, and pathogenesis. These proteins, and protein assemblies, such as pili, are typically long and thin yet must withstand high levels of mechanical stress and proteolytic attack. The recent discovery of intramolecular isopeptide bond cross-links, formed autocatalytically, in the pili from Streptococcus pyogenes has highlighted the role that such cross-links can play in stabilizing such structures. We have investigated a putative cell-surface adhesin from Clostridium perfringens comprising an N-terminal adhesin domain followed by 11 repeat domains. The crystal structure of a two-domain fragment shows that each domain has an IgG-like fold and contains an unprecedented ester bond joining Thr and Gln side chains. MS confirms the presence of these bonds. We show that the bonds form through an autocatalytic intramolecular reaction catalyzed by an adjacent His residue in a serine protease-like mechanism. Two buried acidic residues assist in the reaction. By mutagenesis, we show that loss of the ester bond reduces the thermal stability drastically and increases susceptibility to proteolysis. As in pilin domains, the bonds are placed at a strategic position joining the first and last strands, even though the Ig fold type differs. Bioinformatic analysis suggests that similar domains and ester bond cross-links are widespread in Gram-positive bacterial adhesins.