Role of the amino latch of staphylococcal α-hemolysin in pore formation -: A co-operative interaction between the N terminus and position 217
Role of the amino latch of staphylococcal α-hemolysin in pore formation -: A co-operative interaction between the N terminus and position 217
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DOI：
10.1074/jbc.m510841200
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发表时间：
2006-01-27
影响因子：
4.8
通讯作者：
Bayley, H
中科院分区：
文献类型：
--
作者：
Jayasinghe, L;Miles, G;Bayley, H
葡萄球菌α溶血素（α HL）是一种β桶孔形成毒素，由细菌分泌为水溶性单体蛋白。在与易感细胞结合后，α - HL通过一个无活性的预孔组装形成一个充满水的同七聚体跨膜孔。α HL的N端在完全组装的孔隙的晶体结构中形成相邻亚基之间的锁存器，被认为在pre - pore - to - pore转换中起着至关重要的作用。例如，两个n端残基的删除产生了一个完全失活的蛋白质，在准备阶段在组装中被阻止。在目前的研究中，我们用一组全面的截断突变体重新检查了组装。令人惊讶的是，我们发现截断多达17个氨基酸后，α HL形成功能性孔的能力减弱，但仍然很大。然后，我们发现突变Ser(217) -> Asn存在于我们的原始截断集中，而不存在于新的截断集中，它促进了N端截断时的完全失活。因此，α HL的N端并不像之前认为的那样对pre - pore to - pore的转化至关重要。残基217参与了组装过程，并且必须在孔形成的最后一步与远端N端间接相互作用。此外，我们提供的证据表明，完整的N端可以防止α HL单体在溶液中过早寡聚。
Staphylococcal alpha-hemolysin (alpha HL) is a beta barrel pore-forming toxin that is secreted by the bacterium as a water-soluble monomeric protein. Upon binding to susceptible cells, alpha HL assembles via an inactive prepore to form a water-filled homoheptameric transmembrane pore. The N terminus of alpha HL, which in the crystal structure of the fully assembled pore forms a latch between adjacent subunits, has been thought to play a vital role in the prepore to pore conversion. For example, the deletion of two N-terminal residues produced a completely inactive protein that was arrested in assembly at the prepore stage. In the present study, we have re-examined assembly with a comprehensive set of truncation mutants. Surprisingly, we found that after truncation of up to 17 amino acids, the ability of alpha HL to form functional pores was diminished, but still substantial. We then discovered that the mutation Ser(217) -> Asn, which was present in our original set of truncations but not in the new ones, promotes complete inactivation upon truncation of the N terminus. Therefore, the N terminus of alpha HL cannot be critical for the prepore to pore transformation as previously thought. Residue 217 is involved in the assembly process and must interact indirectly with the distant N terminus during the last step in pore formation. In addition, we provide evidence that an intact N terminus prevents the premature oligomerization of alpha HL monomers in solution.