Structure-Activity Relationship of Gelatinase Biosynthesis-Activating Pheromone of Enterococcus faecalis
Structure-Activity Relationship of Gelatinase Biosynthesis-Activating Pheromone of Enterococcus faecalis
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DOI：
10.1128/jb.01029-08
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发表时间：
2009-01-15
影响因子：
3.2
通讯作者：
Nakayama, Jiro
中科院分区：
文献类型：
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作者：
Nishiguchi, Kenzo;Nagata, Koji;Nakayama, Jiro
粪肠球菌（Enterococcus faecalis）中与致病性相关的胞外蛋白酶（即明胶酶和丝氨酸蛋白酶）的表达受一种称为明胶酶生物合成激活信息素（gelatinase biosynthesis-activating pheromone，GBAP）的自诱导肽介导的群体感应系统正调控。GBAP是含有内酯键的11个氨基酸残基的环状肽。为了研究GBAP的构效关系，我们合成了一系列GBAP类似物，并通过明胶酶诱导试验和新开发的受体结合试验评估其活性，其中荧光标记的肽结合到Fsrc过表达的乳酸乳球菌细胞表面上，通过荧光显微镜观察并通过使用荧光光度计定量。对GBAP的丙氨酸扫描分析表明，整个环区参与了GBAP激动剂活性，而尾区的侧链没有被严格识别。Phe（7）或Trp（10）的丙氨酸取代几乎消除了它们的受体结合能力和GBAP激动剂活性，表明这两个芳香族侧链强烈参与受体相互作用和活化。此外，Trp（10）取代天然和非天然的芳香族氨基酸，除了五氟苯丙氨酸，没有引起激动剂活性的损失。这表明了由芳香环表面上的π电子云产生的负静电势的重要性。用核磁共振波谱对GBAP进行结构分析，发现其环状区域呈发夹状折叠，并紧密堆积成一个紧凑的形式。Trp（10）的侧链部分地埋在核心结构中，有助于致密形式的稳定，而Phe（7）的侧链从核心结构延伸到溶剂中，可能直接参与受体结合。
The expression of pathogenicity-related extracellular proteases, namely, gelatinase and serine protease, in Enterococcus faecalis is positively regulated by a quorum-sensing system mediated by an autoinducing peptide called gelatinase biosynthesis-activating pheromone (GBAP). GBAP is an 11-amino-acid-residue cyclic peptide containing a lactone linkage. To study the structure-activity relationship of GBAP, we synthesized a series of GBAP analogues and evaluated their activities by a gelatinase-inducing assay and newly developed receptor-binding assays in which fluorescence-labeled peptides bound onto the FsrC-overexpressing Lactococcus lactis cell surface were observed by fluorescent microscopy and quantified by using a fluorophotometer. Alanine-scanning analysis of GBAP showed that the entire ring region was involved in the GBAP agonist activity, while side chains of the tail region were not strictly recognized. The alanine substitution of Phe(7) or Trp(10) almost abolished their receptor-binding abilities and GBAP agonist activities, suggesting that these two aromatic side chains are strongly involved in receptor interaction and activation. Furthermore, the Trp(10) substitution with natural and unnatural aromatic amino acids, except pentafluorophenylalanine, caused no loss of agonist activity. This suggested the importance of a negative electrostatic potential created by an pi-electron cloud on the aromatic ring surface. Structural analysis of GBAP with nuclear magnetic resonance spectroscopy revealed that the ring region adopted a hairpin-like fold and was tightly packed into a compact form. The side chain of Trp(10) was partially buried in the core structure, contributing to the stabilization of the compact form, while that of Phe(7) was extended from the core structure into the solvent and was probably directly involved in receptor binding.