C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates
C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates
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DOI：
10.1046/j.1365-2958.2002.02889.x
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发表时间：
2002-04-01
影响因子：
3.6
通讯作者：
Scherer, S
中科院分区：
文献类型：
--
作者：
Loessner, MJ;Kramer, K;Scherer, S
单核细胞增生性李斯特菌噬菌体内切酶Ply118和Ply500具有独特的酶活性，能在病毒增殖完成时特异性地降解李斯特菌细胞，从而释放出后代噬菌体。为了确定这些酶的裂解特异性的分子基础，我们阐明了它们的结构域结构，并研究了它们无关和唯一的C末端细胞壁结合结构域(CBD)的功能。缺失突变体分析表明，这两个结构域都是裂解活性所必需的。CBDS与绿色荧光蛋白(GFP)的融合表明，Ply500的C端140个氨基酸和Ply118的C端182个残基是将Murein水解酶定向到细菌细胞壁的必要条件和充分条件。CBD500能够将GFP靶向4、5和6群李斯特氏菌的细胞表面，使整个细胞表面染色均匀。相反，CBD118杂交种与配体的结合主要存在于隔区和细胞极点，但仅存在于1/2、3和7血清型的细胞上。与表面碳水化合物配体的非共价结合以一种快速、饱和依赖的方式发生。我们分别测量了CBD118和CBD500的4×10(4)和8×10(4)结合位点。表面等离子体共振分析揭示了CBD-配体相互作用的意外高分子亲和力常数，对应于纳摩尔亲和力。综上所述，我们发现CBD负责将噬菌体内毒素定向到它们的底物上，并赋予蛋白质识别特异性。由于CBD序列不包含重复序列，并且缺乏所有已知的将蛋白质锚定到细菌细胞上的序列基序，我们得出结论，它们使用独特的结构基序与革兰氏阳性细菌表面特异性结合。
Listeria monocytogenes phage endolysins Ply118 and Ply500 share a unique enzymatic activity and specifically hydrolyse Listeria cells at the completion of virus multiplication in order to release progeny phage. With the aim of determining the molecular basis for the lytic specificity of these enzymes, we have elucidated their domain structure and examined the function of their unrelated and unique C-terminal cell wall binding domains (CBDs). Analysis of deletion mutants showed that both domains are needed for lytic activity. Fusions of CBDs with green fluorescent protein (GFP) demonstrated that the C-terminal 140 amino acids of Ply500 and the C-terminal 182 residues of Ply118 are necessary and sufficient to direct the murein hydrolases to the bacterial cell wall. CBD500 was able to target GFP to the surface of Listeria cells belonging to serovar groups 4, 5 and 6, resulting in an even staining of the entire cell surface. In contrast, the CBD118 hybrid bound to a ligand predominantly present at septal regions and cell poles, but only on cells of serovars 1/2, 3 and 7. Non-covalent binding to surface carbohydrate ligands occurred in a rapid, saturation-dependent manner. We measured 4 x 10(4) and 8 x 10(4) binding sites for CBD118 and CBD500 respectively. Surface plasmon resonance analysis revealed unexpected high molecular affinity constants for the CBD-ligand interactions, corresponding to nanomolar affinities. In conclusion, we show that the CBDs are responsible for targeting the phage endolysins to their substrates and function to confer recognition specificity on the proteins. As the CBD sequences contain no repeats and lack all known sequence motifs for anchoring of proteins to the bacterial cell, we conclude that they use unique structural motifs for specific association with the surface of Gram-positive bacteria.