Residues involved in the pore-forming activity of the Clostridium perfringens iota toxin
Residues involved in the pore-forming activity of the Clostridium perfringens iota toxin
复制标题
DOI：
10.1111/cmi.12366
复制
发表时间：
2015-02-01
影响因子：
3.4
通讯作者：
Popoff, Michel R.
中科院分区：
文献类型：
--
作者：
Knapp, Oliver;Maier, Elke;Popoff, Michel R.
产气荚膜梭菌毒素是一种由酶（Ia）和结合物（Ib）组成的二元毒素。Ib在脂质双分子层中形成通道，并介导Ia向靶细胞的转运。在这里，我们发现Ib残基334-359包含一个保守的疏水和亲水交替残基模式，形成两个两亲性β链，参与膜插入和通道形成。这段氨基酸显示出与产气荚膜荚膜杆菌毒素的β孔形成结构域的显著结构和功能相似性。两种两亲性β -链中的一些突变影响了孔形成、单通道电导和离子选择性（S339E-S341E, Q345H N346E），证实了它们参与通道形成。Ib的F454对应于炭疽保护性抗原的Phi-clamp F427和C2II二元毒素的F428。突变F454A导致细胞毒性丧失，单通道电导显著增加（在1 M KCl中为500 pS，而在1 M KCl中为85 pS），阳离子选择性略有下降，表明ph -clamp高度保守，对二元毒素活性至关重要。相比之下，突变体Q367D、N430D、L443E对Ib性质没有或只有轻微影响，而T360I、T360A和T360W对离子选择性和单通道电导产生了显著影响，表明低聚物结构受到了严重干扰。这表明，至少在iota毒素家族中，T360在孔隙组织中具有结构性作用。此外，在通道（S339E-S341E）或前庭（Q367D， N430D和L443E）中引入带电残基对氯喹和Ia的结合几乎没有影响，而F454A， T360I， T360A和T360W则强烈降低了氯喹和Ia对Ib的亲和力。这些结果支持前庭内不同的残基与氯喹和Ia相互作用或负责通道结构，而通道衬里氨基酸的作用不太重要。
Clostridium perfringens iota toxin is a binary toxin that is organized into enzyme (Ia) and binding (Ib) components. Ib forms channels in lipid bilayers and mediates the transport of Ia into the target cells. Here we show that Ib residues 334-359 contain a conserved pattern of alternating hydrophobic and hydrophilic residues forming two amphipathic beta-strands involved in membrane insertion and channel formation. This stretch of amino acids shows remarkable structural and functional analogies with the beta-pore-forming domain of C. perfringens epsilon toxin. Several mutations within the two amphipathic beta-strands affected pore formation, single-channel conductance and ion selectivity (S339E-S341E, Q345H N346E) confirming their involvement in channel formation. F454 of Ib corresponds to the Phi-clamp F427 of anthrax protective antigen and F428 of C2II binary toxins. The mutation F454A resulted in a loss of cytotoxicity and strong increase in single-channel conductance (500 pS as compared with 85 pS in 1 M KCl) with a slight decrease in cation selectivity, indicating that the Phi-clamp is highly conserved and crucial for binary toxin activity. In contrast, the mutants Q367D, N430D, L443E had no or only minor effects on Ib properties, while T360I, T360A and T360W caused a dramatic effect on ion selectivity and single-channel conductance, indicating gross disturbance of the oligomer structure. This suggests that, at least in the iota toxin family, T360 has a structural role in the pore organization. Moreover, introduction of charged residues within the channel (S339E-S341E) or in the vestibule (Q367D, N430D and L443E) had virtually no effect on chloroquine or Ia binding, whereas F454A, T360I, T360A and T360W strongly decreased the chloroquine and Ia affinity to Ib. These results support that distinct residues within the vestibule interact with chloroquine and Ia or are responsible for channel structure, while the channel lining amino acids play a less important role.