The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor.
The CD27L and CTP1L endolysins targeting Clostridia contain a built-in trigger and release factor.
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DOI：
10.1371/journal.ppat.1004228
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发表时间：
2014-07
期刊：
影响因子：
6.7
通讯作者：
Meijers R
中科院分区：
文献类型：
--
作者：
Dunne M;Mertens HD;Garefalaki V;Jeffries CM;Thompson A;Lemke EA;Svergun DI;Mayer MJ;Narbad A;Meijers R
噬菌体Φ CD 27能够裂解艰难梭菌，这是一种致病性细菌，是医院感染的主要原因。重组CD 27 L内溶素裂解C. difficile在体外，并代表了一个有前途的替代作为一种药物。为了更好地理解溶解机制，我们已经确定了CD 27 L内溶素的自蛋白水解片段的晶体结构。该结构覆盖了内溶素的C-末端结构域，并且代表了在靶向梭菌属细菌的许多溶素中鉴定的新折叠。该结构表明内溶素裂解发生在连接催化结构域与C-末端结构域的接头的茎部。我们还解决了靶向C的CTP 1 L内溶素的缓慢切割突变体的C末端结构域的晶体结构。酪丁酸菌两种内溶素的晶体结构中观察到两种不同的二聚化模式，尽管结构域之间的序列同一性仅为22%。通过直角光散射、小角X射线散射和使用交联氨基酸对苯甲酰基-L-苯丙氨酸（pBpa）的交联实验，验证了溶液中全长蛋白质存在二聚体。对二聚体界面有贡献的残基的突变表明二聚化模式和自裂解机制之间存在联系。我们表明，对于CTP 1 L内溶素，存在与裂解效率成比例的裂解效率的降低。我们提出了一个模型内溶素触发，其中扩展的二聚体呈现非活性状态，和开关的并排二聚体触发裂解的C-末端结构域。这导致内溶素的催化部分的释放，使得能够有效消化细菌细胞壁。 艰难梭菌感染是医院获得性腹泻的常见原因，特别是在老年患者中，在广谱抗生素治疗后。噬菌体疗法可以提供一种替代治疗方法，但更好地了解裂解细菌细胞的病毒成分是必要的。在这里，我们报告了两个内溶素的激活噬菌体裂解梭菌。用X-射线晶体衍射法测定了两种内溶素的自蛋白水解片段的结构。基于这些结构，我们引入了影响内溶素的酶促部分的自溶裂解的突变，并且我们表明两种寡聚状态对裂解机制有影响。此外，当一种内溶素的自裂解被抑制时，裂解活性受到影响。我们认为，裂解和寡聚化是相互联系的，它们为内溶素提供了一种触发和释放机制，从而导致激活。触发和释放因子的鉴定可能不仅与梭菌内溶素相关，而且可能是触发许多噬菌体内溶素的重要因素。对这种激活机制的更全面的理解将有助于设计具有更有效治疗潜力的重组内溶素或噬菌体。
The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we have determined the crystal structure of an autoproteolytic fragment of the CD27L endolysin. The structure covers the C-terminal domain of the endolysin, and represents a novel fold that is identified in a number of lysins that target Clostridia bacteria. The structure indicates endolysin cleavage occurs at the stem of the linker connecting the catalytic domain with the C-terminal domain. We also solved the crystal structure of the C-terminal domain of a slow cleaving mutant of the CTP1L endolysin that targets C. tyrobutyricum. Two distinct dimerization modes are observed in the crystal structures for both endolysins, despite a sequence identity of only 22% between the domains. The dimers are validated to be present for the full length protein in solution by right angle light scattering, small angle X-ray scattering and cross-linking experiments using the cross-linking amino acid p-benzoyl-L-phenylalanine (pBpa). Mutagenesis on residues contributing to the dimer interfaces indicates that there is a link between the dimerization modes and the autocleavage mechanism. We show that for the CTP1L endolysin, there is a reduction in lysis efficiency that is proportional to the cleavage efficiency. We propose a model for endolysin triggering, where the extended dimer presents the inactive state, and a switch to the side-by-side dimer triggers the cleavage of the C-terminal domain. This leads to the release of the catalytic portion of the endolysin, enabling the efficient digestion of the bacterial cell wall. Clostridium difficile infection is a common cause of hospital-acquired diarrhea, following broad-spectrum antibiotic treatment particularly in elderly patients. Bacteriophage therapy could provide an alternative treatment, but a better understanding of the viral components that lyse the bacterial cell is necessary. Here, we report on the activation of two endolysins from bacteriophages that lyse Clostridia. The structures of autoproteolytic fragments of two endolysins were determined by X-ray crystallography. Based on the structures, we introduced mutations that affect the autolytic cleavage of the enzymatic portion of the endolysins, and we show that two oligomeric states have an effect on the cleavage mechanism. Moreover, the lysis activity is affected when autocleavage is inhibited for one endolysin. We propose that the cleavage and oligomerization are linked, and they provide the endolysin with a trigger and release mechanism that leads to activation. The identification of a trigger and release factor may not only be relevant to Clostridia endolysins, but could be an important factor in the triggering of many bacteriophage endolysins. A fuller understanding of this activation mechanism will help in the design of recombinant endolysins or bacteriophages with a more efficient therapeutic potential.
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DOI：
10.1107/s0907444911007232
发表时间：
2011-04
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Krissinel E
通讯作者：
Krissinel E
影响因子：
4.8
作者：
Low, Lieh Yoon;Yang, Chen;Liddington, Robert
通讯作者：
Liddington, Robert
影响因子：
6.1
作者：
Konarev, PV;Volkov, VV;Svergun, DI
通讯作者：
Svergun, DI
影响因子：
4.4
作者：
Mayer, Melinda J.;Gasson, Michael J.;Narbad, Arjan
通讯作者：
Narbad, Arjan
DOI：
10.1107/s0907444909042073
发表时间：
2010-01
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Chen VB;Arendall WB 3rd;Headd JJ;Keedy DA;Immormino RM;Kapral GJ;Murray LW;Richardson JS;Richardson DC
通讯作者：
Richardson DC