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首页> 外文期刊>Journal of bacteriology >Genetic and Biochemical Analysis of Dimer and Oligomer Interactions of the λ S Holin
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Genetic and Biochemical Analysis of Dimer and Oligomer Interactions of the λ S Holin

机译：λS Holin的二聚体和低聚物相互作用的遗传和生化分析

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摘要

Bacteriophage λ uses a holin-endolysin system for host cell lysis. R, the endolysin, has muralytic activity. S, the holin, is a small membrane protein that permeabilizes the inner membrane at a precisely scheduled time after infection and allows the endolysin access to its substrate, resulting in host cell lysis. λ S has a single cysteine at position 51 that can be replaced by a serine without loss of the holin function. A collection of 27 single-cysteine products of alleles created from λ SC51S were tested for holin function. Most of the single-cysteine variants retained the ability to support lysis. Mutations with the most defective phenotype clustered in the first two hydrophobic transmembrane domains. Several lines of evidence indicate that S forms an oligomeric structure in the inner membrane. Here we show that oligomerization does not depend on disulfide bridge formation, since the cysteineless SC51S(i) is functional as a holin and (ii) shows the same oligomerization pattern as the parental S protein. In contrast, the lysis-defective SA52V mutant dimerizes but does not form cross-linkable oligomers. Again, dimerization does not depend on the natural cysteine, since the cysteineless lysis-defective SA52V/C51S is found in dimers after treatment of the membrane with a cross-linking agent. Furthermore, under oxidative conditions, dimerization via the natural cysteine is very efficient for SA52V. Both SA52V (dominant negative) and SA48V(antidominant) interact with the parental S protein, as judged by oxidative disulfide bridge formation. Thus, productive and unproductive heterodimer formation between the parental protein and the mutants SA52V and SA48V, respectively, may account for the dominant and antidominant lysis phenotypes. Examination of oxidative dimer formation between S variants with single cysteines in the hydrophobic core of the second membrane-spanning domain revealed that positions 48 and 51 are on a dimer interface. These results are discussed in terms of a three-step model leading to S-dependent hole formation in the inner membrane.
机译：噬菌体λ使用holin-内溶素系统进行宿主细胞裂解。 R，内溶素，具有溶胞活性。 S，holin，是一种小膜蛋白，在感染后的精确预定时间透化内膜，并允许内溶素进入其底物，从而导致宿主细胞裂解。 λS在第51位具有一个半胱氨酸，可以被丝氨酸取代而不损失holin功能。测试了从λS C51S 创建的等位基因的27个半胱氨酸单半胱氨酸产物的集合。大多数单半胱氨酸变体保留了支持裂解的能力。具有最缺陷表型的突变聚集在前两个疏水跨膜结构域中。几条证据表明，S在内膜中形成寡聚结构。在这里，我们显示寡聚化不依赖于二硫键的形成，因为无半胱氨酸的S （i）可以作为holin起作用，并且（ii）表现出与亲代S蛋白相同的寡聚化模式。相比之下，裂解缺陷型S A52V 突变体可二聚但不会形成可交联的低聚物。同样，二聚化不依赖于天然半胱氨酸，因为在用交联剂处理膜后，在二聚体中发现了无半胱氨酸的裂解缺陷型S 。此外，在氧化条件下，天然半胱氨酸的二聚作用对于S A52V 非常有效。通过氧化二硫键形成判断，S A52V （显性阴性）和S A48V （显性）均与亲本S蛋白相互作用。因此，亲本蛋白与突变体S A52V 和S A48V 之间的生产性和非生产性异源二聚体形成可能解释了显性和显性裂解表型。检查第二个跨膜结构域的疏水核心中具有半胱氨酸的S半胱氨酸之间的氧化二聚体形成发现，位置48和51在二聚体界面上。根据导致内膜中S依赖性孔形成的三步模型讨论了这些结果。

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