Membrane translocation of charged residues at the tips of hydrophobic helices in the T domain of diphtheria toxin
Membrane translocation of charged residues at the tips of hydrophobic helices in the T domain of diphtheria toxin
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DOI：
10.1021/bi981576s
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发表时间：
1999-01-19
期刊：
影响因子：
2.9
通讯作者：
London, E
中科院分区：
文献类型：
--
作者：
Ren, JH;Sharpe, JC;London, E
白喉毒素的T结构域的低pH触发的膜插入是毒素的C结构域在体内跨膜易位的关键步骤。我们以前建立了T结构域可以与两种不同构象的膜相互作用，一种是TH 8/TH 9螺旋发夹靠近双层表面，另一种是它插入更深，似乎是transmembraneous。由于在低pH下质子化，残基E349和D352上的电荷损失已被认为是跨膜插入的关键步骤，因为它们在连接TH 8和TH 9的环内，并且在跨膜插入时必须穿过膜。在本报告中，通过测量双取代E349 K/D352 K对TH 8/TH 9发夹构象的影响，通过连接到TH 9的荧光基团，检查了这些残基的作用。在pH 4.5时，相对于在349和352处具有野生型残基的T结构域，存在E349 K/D352 K突变体的TH 8/TH 9的浅插入。此外，相对于野生型，用E349 K/D352 K突变体获得更小和/或更少的孔。另一方面，高的T结构域浓度，或进一步降低pH值，允许跨膜插入的野生型和349 K/352 K突变体，以及诱导更大和/或更多的孔。此外，跨膜插入过程是快速的毛皮的突变体和野生型。这表明突变体具有形成类似于野生型T结构域的跨膜结构的能力，因此，在蛋白质的膜穿透区域中引入带电基团不会对跨膜运动引入不可逾越的障碍，T结构域形成跨膜构象的能力与孔之间的联系表明，这些突变在抑制孔形成中的作用可能与T结构域的突变有关。可能部分是由于无法正确插入。此外，观察到降低的pH允许349 K/352 K突变体深度插入，表明存在除E349和D352之外的残基，其质子化促进跨膜插入。
The low pH triggered membrane insertion of the T domain of diphtheria toxin is a critical step in the translocation of the C domain of the toxin across membranes in vivo. We previously established that the T domain can interact with membranes in two distinct conformations, one in which the TH8/TH9 helical hairpin lies close to the bilayer surface and a second in which it inserts more deeply and appears to be transmembraneous. The loss of charge on residues E349 and D352 due to protonation at low pH has been proposed to be a critical step in transmembrane insertion, because they are within a loop connecting TH8 and TH9, and must cross the membrane upon transmembrane insertion. In this report, the role of these residues was examined by measuring the effect of the double substitution E349K/D352K on the conformation of the TH8/TH9 hairpin through a fluorescent group attached to TH9. At pH 4.5, there was shallower insertion of TH8/TH9 of the E349K/D352K mutant relative to T domain with wild-type residues at 349 and 352. In addition, smaller and/or fewer pores were obtained with the E349K/D352K mutant relative to the wild-type. On the other hand, high T domain concentrations, or further decreasing pH, allowed transmembrane insertion of both the wild-type and the 349K/352K mutant as well as induction of larger and/or more numerous pores. Furthermore, the transmembrane insertion process was rapid fur both the mutant and wild-type. This shows that the mutant has the capacity to form a transmembrane structure similar to that of the wild-type T domain and, thus, that introduction of charged groups in membrane-penetrating regions of a protein does not introduce an insurmountable barrier to transmembrane movement, The linkage between the ability of the T domain to form the transmembrane conformation and pores suggests that the effects of these mutations in inhibiting pore formation are likely to partly result from the inability to insert properly. Additionally, the observation that decreasing pH allows the 349K/352K mutant to insert deeply indicates that there are residues other than E349 and D352 whose protonation promotes transmembrane insertion.