摘要

Zn-依赖性膜定位蛋白酶YvjB先前已被证明可用作LsbB的靶受体，其是无II型无前导乳酸球菌细菌素。尽管yvjB在乳球菌属中高度保守，但细菌素似乎仅对亚种乳酸乳球菌亚种有活性。球菌。对敏感菌株（YvjBMN）和耐药菌株（YvjBMG）的YvjB蛋白质的比较分析表明，它们在31个位置上彼此不同。在这项研究中，我们应用定点突变和进行定向结合研究，以提供生化证据表明LsbB与易感细胞中YvjB的第三跨膜螺旋相互作用。 LsbB和YvjB蛋白的定点突变表明某些氨基酸和LsbB的长度与细菌素活性有关，最可能是通过这两种蛋白的适当相互作用;负责活性的LsbB中的必需氨基酸是色氨酸（Trp25）和末端丙氨酸（Ala30）。还显示Trp25和末端丙氨酸之间的距离对于LsbB活性是至关重要的。 YvjB中与LsbB相互作用的关键区域是第三个跨膜螺旋的开始，特别是氨基酸酪氨酸（Tyr356）和丙氨酸（Ala353）。体外实验表明，LsbB可与YvjBMN和YvjBMG相互作用，但与YvjBMG相互作用的强度明显较小。免疫荧光标记抗体的体内实验表明，LsbB只与携带YvjBMN的细胞特异性相互作用。

The Zn-dependent membrane-located protease YvjB has previously been shown to serve as a target receptor for LsbB, a class II leaderless lactococcal bacteriocin. Although yvjB is highly conserved in the genus Lactococcus, the bacteriocin appears to be active only against the subspecies L. lactis subsp. lactis. Comparative analysis of the YvjB proteins of a sensitive strain (YvjBMN) and a resistant strain (YvjBMG) showed that they differ from each other in 31 positions. In this study, we applied site-directed mutagenesis and performed directed binding studies to provide biochemical evidence that LsbB interacts with the third transmembrane helix of YvjB in susceptible cells. The site-directed mutagenesis of LsbB and YvjB proteins showed that certain amino acids and the length of LsbB are responsible for the bacteriocin activity, most probably through adequate interaction of these two proteins; the essential amino acids in LsbB responsible for the activity are tryptophan (Trp25) and terminal alanine (Ala30). It was also shown that the distance between Trp25 and terminal alanine is crucial for LsbB activity. The crucial region in YvjB for the interaction with LsbB is the beginning of the third transmembrane helix, particularly amino acids tyrosine (Tyr356) and alanine (Ala353). In vitro experiments showed that LsbB could interact with both YvjBMNand YvjBMG, but the strength of interaction is significantly less with YvjBMG. In vivoexperiments with immunofluorescently labeled antibody demonstrated that LsbB specifically interacts only with cells carrying YvjBMN.

http://aem.asm.org/content/82/17/5364.short