Mutational analysis of the role of tryptophan residues in an antimicrobial peptide
Mutational analysis of the role of tryptophan residues in an antimicrobial peptide
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DOI：
10.1021/bi025856q
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发表时间：
2002-07-30
期刊：
影响因子：
2.9
通讯作者：
Nissen-Meyer, J
中科院分区：
文献类型：
--
作者：
Fimland, G;Eijsink, VGH;Nissen-Meyer, J
抗菌肽是由乳酸菌产生的细菌素（IIa类细菌素）类细菌素家族的一种，含有几种高度保守的色氨酸残基。由于膜蛋白中的色氨酸残基通常位于膜-水界面，我们假设细菌素中的色氨酸残基可能是膜结合肽结构和抗微生物活性的重要决定因素。为了验证这一假设，研究了43个残基的pediocin-like bactericin sakacin P中的3个色氨酸残基（Trp18、Trp33和Trp41）突变的影响。trp18和Trp33位于两亲体α -螺旋的两端，而Trp41位于非结构化c端尾部附近。用疏水残基Len和Phe取代Trp33对活性的影响微乎其微，而用极性更强的Tyr和Arg取代Trp33则分别使活性降低了10-20倍和500-1000倍，这表明Trp33和螺旋的c端部分与膜的疏水核心相互作用。Trp18和Trp41的任何突变都会降低活性，表明这两个残基发挥着独特的作用。Trp18和Trp41与膜-水界面相互作用的结果表明，Trp18和Trp41与膜-水界面相互作用。建议的三个Trp残基位置与螺旋和c端尾部形成发夹状结构的结构模型相容，该结构模型使Trp18和Trp41在界面上彼此靠近，而Trp33位于膜的疏水核心。事实上，W18L和W41L突变的有害影响可以通过在残基24和残基44之间引入二硫桥来稳定发夹状结构来克服。这些结果为建立一个精细的pediocin样细菌素结构模型提供了基础，并突出了色氨酸残基在膜相互作用肽中发挥的独特作用。
Antimicrobial peptides belonging to the pediocin-like family of bacteriocins (class IIa bacteriocins) produced by lactic acid bacteria contain several tryptophan residues that are highly conserved. Since tryptophan residues in membrane proteins are often positioned in the membrane-water interface, we hypothesized that Trp residues in bacteriocins could be important determinants of the structure of membrane-bound peptides and of anti-microbial activity. To test this hypothesis, the effects of mutating each of the 3 tryptophan residues (Trp18, Trp33, and Trp41) in the 43-residue pediocin-like bacteriocin sakacin P were studied. Trp 18 and Trp33 are located at each end of an amphihilic alpha-helix, whereas Trp41 is near the end of an unstructured C-terminal tail. Replacement of Trp33 with the hydrophobic residues Len and Phe had marginal effects on activity, whereas replacement with the more polar Tyr and Arg reduced activity 10-20 and 500-1000 times, respectively, indicating that Trp33 and the C-terminal part of the helix interact with the hydrophobic core of the membrane. Any mutation of Trp18 and Trp41 reduced activity, indicating that these two residues play unique roles. Substitutions with other aromatic residues were the least deleterious, indicating that both Trp18 and Trp41 interact with the membrane-water interface. The suggested locations of the three Trp residues are compatible with a structural model in which the helix and the C-terminal tail form a hairpin-like structure, bringing Trp18 and Trp41 close to each other in the interface, and placing Trp33 in the hydrophobic core of the membrane. Indeed, the deleterious effect of the W18L and W41L mutations could be overcome by stabilizing the hairpin-like structure by introduction of a disulfide bridge between residues 24 and 44. These results provide a basis for a refined structural model of pediocin-like bacteriocins and highlight the unique role that tryptophan residues can play in membrane-interacting peptides.