Dementiev Alexey, Board Jason, Sitaram Anand, Hey Timothy, Kelker Matthew S, Xu Xiaoping, Hu Yan, Vidal-Quist Cristian, Chikwana Vimbai, Griffin Samantha, McCaskill David, Wang Nick X, Hung Shao-Ching, Chan Michael K, Lee Marianne M, Hughes Jessica, Wegener Alice, Aroian Raffi V, Narva Kenneth E, Berry Colin
Shamrock Structures LLC, Woodridge, IL, USA.
Cardiff School of Biosciences, Cardiff University, Park Place, Cardiff, CF15 8FA, UK.
BMC Biol. 2016 Aug 30;14(1):71. doi: 10.1186/s12915-016-0295-9.
The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action.
The two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action.
Cry6 proteins are members of the alpha helical pore-forming toxins - a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities.
苏云金芽孢杆菌的Cry6蛋白家族代表了一组强大的毒素，在控制鞘翅目昆虫以及对农业具有重要意义的线虫寄生虫方面具有巨大的应用潜力。这些蛋白在一级序列水平上与其他杀虫毒素无关，并且迄今为止对这些蛋白的结构和功能研究甚少。这阻碍了我们对这些毒素及其作用方式的理解，以及我们为了自身利益操纵这些蛋白以改变其活性的能力。为了增进我们对其作用方式的理解并促进这些蛋白的进一步开发，我们确定了原毒素形式和胰蛋白酶激活形式的Cry6Aa的结构，并证明了其成孔作用机制。
毒素的两种形式分别解析到2.7 Å和2.0 Å，并且显示出非常相似的结构。Cry6Aa与一类已知的成孔毒素具有结构同源性，包括来自大肠杆菌的溶血素E以及两种蜡样芽孢杆菌蛋白：溶血毒素HblB和非溶血毒素的NheA组分（pfam05791）。与该家族的其他成员相比，Cry6Aa还表现出非典型特征，包括内部重复序列和主要α螺旋内的小环区域。发现胰蛋白酶处理导致一些内部序列丢失，而C末端区域仍通过二硫键与毒素的主要核心相连。基于Cry6Aa与其他毒素的结构相似性，探究了毒素的作用机制，并证明了其在秀丽隐杆线虫体内形成孔的能力。还产生了一个无毒突变体，这与所提出的成孔作用方式一致。
Cry6蛋白是α螺旋成孔毒素家族的成员——这是苏云金芽孢杆菌的Cry毒素中以前未被识别的一种结构类别，代表了杀线虫和杀虫蛋白的一种新范例。Cry6Aa的结构和成孔作用机制的阐明现在为更详细地分析毒素特异性以及开发具有新活性的新毒素变体开辟了道路。
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