Kazazic Maja, Nissen-Meyer Jon, Fimland Gunnar
Department of Biochemistry, University of Oslo, Post Box 1041, Blindern, 0316 Oslo, Norway1.
Microbiology (Reading). 2002 Jul;148(Pt 7):2019-2027. doi: 10.1099/00221287-148-7-2019.
The significance of charged residues for the target-cell binding, potency and specificity of pediocin-like bacteriocins has been studied by site-directed mutagenesis of sakacin P. Most of the charged residues are located in the N-terminal half, which is thought to mediate the initial binding of these bacteriocins to target cells through electrostatic interaction. All the mutated peptides in which the net positive charge was reduced by one (by replacing a charged residue with threonine) exhibited reduced binding to target cells and a 2-15-fold reduction in potency. The least deleterious of these mutations was the removal of the positive charge in position 8 (H8T). This mutation was, in fact, less deleterious than the conservative His to Lys mutation, indicating that the positive charge in position 8 per se is not of major importance. Somewhat more deleterious was the removal of positive charges at the N- and C-terminal ends (K1T, K43T). Most deleterious was the elimination of the positive charge at positions 11 and (but to a lesser extent) 12, demonstrating the importance of the cationic patch in the middle of the N-terminal half of pediocin-like bacteriocins. Mutated peptides in which the net positive charge was increased by one were also constructed. Some of these exhibited increased cell binding and a potency that was the same as (44K, i.e. an extra positive charge at the C-terminus), or somewhat greater (T20K) than, that of sakacin P, whereas others (0K, i.e. an extra positive charge at the N-terminus) had reduced potency. Sakacin P contains only one negatively charged residue (Asp17). This negative charge and its orientation in space were crucial for activity, since the Asp to Asn mutation and (especially) the conservative Asp to Glu mutation were deleterious. Mutations that made the peptide less cationic had, overall, less effect on the potency toward the Carnobacterium piscicola strain than on the potency toward the three other strains tested, whereas the opposite was the case for mutations that made the peptide more cationic. Thus, charged residues in the N-terminal half may - apparently via the initial electrostatic binding of the bacteriocin to target cells - influence the target-cell specificity.
通过对片球菌素P进行定点诱变，研究了带电荷残基对类片球菌素的靶细胞结合、效力和特异性的重要性。大多数带电荷残基位于N端的前半部分，人们认为这部分通过静电相互作用介导这些细菌素与靶细胞的初始结合。所有净正电荷减少一个（通过用苏氨酸取代带电荷残基）的突变肽与靶细胞的结合能力均降低，效力降低了2至15倍。这些突变中危害最小的是去除第8位的正电荷（H8T）。事实上，这种突变比保守的组氨酸突变为赖氨酸的突变危害更小，这表明第8位的正电荷本身并不重要。在N端和C端去除正电荷（K1T、K43T）的危害稍大一些。危害最大的是去除第11位和（程度稍轻的）第12位的正电荷，这表明类片球菌素N端前半部分中间的阳离子区很重要。还构建了净正电荷增加一个的突变肽。其中一些表现出与靶细胞结合能力增强，效力与片球菌素P相同（44K，即C端额外增加一个正电荷），或稍强（T20K），而其他一些（0K，即N端额外增加一个正电荷）效力降低。片球菌素P仅含有一个带负电荷的残基（Asp17）。这个负电荷及其在空间中的方向对活性至关重要，因为天冬氨酸突变为天冬酰胺以及（尤其是）保守的天冬氨酸突变为谷氨酸的突变都是有害的。总体而言，使肽的阳离子性降低的突变对嗜鱼肉杆菌菌株的效力影响比对其他三种测试菌株的效力影响小，而使肽的阳离子性增加的突变情况则相反。因此，N端前半部分的带电荷残基可能——显然是通过细菌素与靶细胞的初始静电结合——影响靶细胞特异性。
