Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O:: An α-helical to β-sheet transition identified by fluorescence spectroscopy
Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O:: An α-helical to β-sheet transition identified by fluorescence spectroscopy
复制标题
DOI：
10.1021/bi981452f
复制
发表时间：
1998-10-13
期刊：
影响因子：
2.9
通讯作者：
Tweten, RK
中科院分区：
文献类型：
--
作者：
Shepard, LA;Heuck, AP;Tweten, RK
产气荚膜梭菌产气荚膜梭菌溶素O（PFO或θ-毒素）是一种溶细胞毒素，其与含胆固醇的膜结合，然后自缔合以在双层中自发形成不同大小的水性孔。在这项研究中，已经通过使用荧光染料N，N '-二甲基-N-（碘乙酰基）-N'-（7-硝基苯并-2-氧杂-1，3-二唑基）乙二胺（NBD）的荧光光谱方法的组合在PFO中鉴定了跨膜结构域，所述荧光染料的发射特性对水敏感。PFO在氨基酸K189和N218之间的大部分残基被单个半胱氨酸取代，然后每个半胱氨酸被NBD修饰。然后将每个纯化的NBD标记的PFO结合到膜上，并通过测量其荧光寿命，发射强度和与水性（碘离子）或非水性（氮氧自由基标记的磷脂）猝灭剂的碰撞猝灭来确定探针的环境。寿命和强度测量显示，在膜结合的PFO多肽的该区域中的氨基酸侧链在水性或非水性环境中交替。这种模式表明，这部分膜结合的PFO以反平行β折叠构象跨越膜。这些残基交替暴露于双层的疏水内部，证明了他们的敏感性淬灭连接到磷脂酰基链的氮氧化物部分。因此，残基K189-N218在膜结合的PFO中形成双链的两亲性β-折叠，其在孔和膜之间产生稳定的界面。这个相同的区域在可溶性单体形式的PFO中包装为三个短的α-螺旋，因此，PFO向膜结合寡聚体的胆固醇依赖性转化涉及主要的结构转变，其中三个α-螺旋解折叠形成跨膜两亲性β-折叠。
Clostridium perfringens perfringolysin O (PFO or theta-toxin) is a cytolytic toxin that binds to cholesterol-containing membranes and then self-associates to spontaneously form aqueous pores of varying size in the bilayer. In this study, a membrane-spanning domain has been identified in PFO by a combination of fluorescence spectroscopic methods using the fluorescent dye N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazolyl)ethylenediamine (NBD) whose emission properties are sensitive to water. PFO was substituted with a single cysteine at most of the residues between amino acids K189 and N218, and then each cysteine was modified with NBD. Each purified NBD-labeled PFO was then bound to membranes, and the probe's environment was ascertained by measuring its fluorescence lifetime, emission intensity, and collisional quenching with either aqueous (iodide ions) or nonaqueous (nitroxide-labeled phospholipids) quenchers. Lifetime and intensity measurements revealed that the amino acid side chains in this region of the membrane-bound PFO polypeptide alternated between being in an aqueous or a nonaqueous environment. This pattern indicates that this portion of the membrane-bound PFO spans the membrane in an antiparallel beta-sheet conformation. The alternating exposure of these residues to the hydrophobic interior of the bilayer was demonstrated by their susceptibility to quenching by nitroxide moieties attached to phospholipid acyl chains. Residues K189-N218 therefore form a two-stranded, amphipathic beta-sheet in the membrane-bound PFO that creates a stable interface between the pore and the membrane. This same region packs as three short cr-helices in the soluble, monomeric form of PFO, and therefore, the cholesterol-dependent conversion of PFO to a membrane-bound oligomer involves a major structural transition in which three alpha-helices unfold to form a membrane-spanning amphipathic beta-sheet.