PH-DEPENDENT INSERTION OF PROTEINS INTO MEMBRANES - B-CHAIN MUTATION OF DIPHTHERIA-TOXIN THAT INHIBITS MEMBRANE TRANSLOCATION, GLU-349-]LYS
PH-DEPENDENT INSERTION OF PROTEINS INTO MEMBRANES - B-CHAIN MUTATION OF DIPHTHERIA-TOXIN THAT INHIBITS MEMBRANE TRANSLOCATION, GLU-349-]LYS
复制标题
DOI：
10.1073/pnas.89.13.6202
复制
发表时间：
1992-07-01
影响因子：
11.1
通讯作者：
COLLIER, RJ
中科院分区：
文献类型：
--
作者：
OKEEFE, DO;CABIAUX, V;COLLIER, RJ
为了研究白喉毒素（DT）如何在哺乳动物细胞中经历pH依赖性膜移位，我们分离并表征了该毒素的突变体，这些突变体在酸性pH依赖性杀死大肠杆菌方面存在缺陷。克隆DT分泌到E.大肠杆菌在酸性条件下（接近pH 5.0）通过插入并透化内膜（一种与毒素的ADP-核糖基化活性无关的机制）杀死细菌。DT突变体对E.通过在酸性条件下接种细菌来选择大肠杆菌。CRM 503是通过该方案选择的全长突变体之一，也显示出对哺乳动物细胞的细胞毒性降低。我们将CRM 503的细胞毒性改变追溯到B片段内的Glu-349 -> Lys突变（E349 K），这是三个点突变之一。E349 K突变单独抑制哺乳动物细胞的细胞毒性和膜转位，并抑制E.但不影响酶活性或受体结合。最近确定的DT晶体学模型表明，Glu-349位于连接易位结构域的两个长疏水-α-螺旋的短环内。Glu-349和附近的另外两个酸性残基Asp-352和Glu-362的质子化可以使这些螺旋经历膜插入，并且插入环被转移到双层的相对面。E349 K突变在该位点引入正电荷，预期其抑制膜插入和DT的插入依赖性活性。这些结果表明，质子化的Glu-349和附近的酸性残基可能是重要的触发毒素作用的易位步骤。
To investigate how diphtheria toxin (DT) undergoes pH-dependent membrane translocation in mammalian cells, we have isolated and characterized mutants of the toxin that are defective in acidic-pH-dependent killing of Escherichia coli. Cloned DT secreted to the periplasm of E. coli kills the bacteria under acidic conditions (near pH 5.0) by inserting into and permeabilizing the inner membrane (a mechanism independent of the toxin's ADP-ribosylation activity). Mutant forms of DT with reduced lethality for E. coli were selected by plating the bacteria under acidic conditions. CRM503, one of the full-length mutants selected by this protocol, also showed diminished cytotoxicity for mammalian cells. We traced the altered cytotoxicity of CRM503 to a Glu-349 --> Lys mutation (E349K), one of three point mutations, within the B fragment. The E349K mutation alone inhibited cytoxicity and membrane translocation in mammalian cells and lethality for E. coli but did not affect enzymic activity or receptor binding. The recently determined crystallographic model of DT shows that Glu-349 resides within a short loop connecting two long hydrophobic-alpha-helices of the translocation domain. Protonation of Glu-349 and two other nearby acidic residues, Asp-352 and Glu-362, may enable these helices to undergo membrane insertion and the intervening loop to be transferred to the opposite face of the bilayer. The E349K mutation introduces a positive charge at this site, which would be expected to inhibit membrane insertion and the insertion-dependent activities of DT. These results suggest that protonation of Glu-349 and nearby acidic residues may be important in triggering the translocation step of toxin action.