Benson E L, Huynh P D, Finkelstein A, Collier R J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 1998 Mar 17;37(11):3941-8. doi: 10.1021/bi972657b.
In its activated 63 kDa form, the protective antigen (PA) component of anthrax toxin forms a heptameric prepore, which converts to a pore (channel) in endosomal membranes at low pH and mediates translocation of the toxin's enzymic moieties to the cytosol. It has been proposed that the prepore-to-pore conversion involves a conformational rearrangement of a disordered amphipathic loop (D2L2; residues 302-325), in which loops from the 7 protomers combine to form a transmembrane 14-stranded beta barrel. To test this model, we generated Cys substitutions in 24 consecutive residues of the D2L2 loop, formed channels in artificial bilayers with each mutant, and examined changes in channel conductance after adding the thiol-reactive, bilayer-impermeant reagent methanethiosulfonate ethyltrimethylammonium (MTS-ET) to the trans compartment. The rationale for these experiments is that reaction of MTS-ET with a Cys residue adds a positively charged group and therefore would likely reduce channel conductance if the residue were in the ion-conducting pathway. We found alternating reduction and absence of reduction of conductance in consecutive residues over two stretches (residues 302-311 and 316-325). This pattern is consistent with alternating polar and apolar residues of the two stretches projecting into the pore lumen and into the bilayer, respectively. Residues connecting these two stretches (residues 312-315) were responsive to MTS-ET, consistent with their being in a turn region. Single channels formed by selected mutants (H304C and N306C) showed multiple conductance step changes in response to MTS-ET, consistent with an oligomeric pore. We also found that the binding site for the channel-blocking tetraalkylammonium ions is located cis relative to the inserted D2L2 loops. These findings constitute strong evidence in favor of the model of conversion of the prepore to a 14-stranded beta barrel pore and solidify the foundation for studies to understand the mechanism of translocation by anthrax toxin.
炭疽毒素的保护性抗原（PA）成分在其活化的63 kDa形式下形成七聚体前体孔，该前体孔在低pH值下会在内体膜中转化为孔（通道），并介导毒素的酶部分转运至细胞质溶胶。有人提出，前体孔到孔的转化涉及无序两亲性环（D2L2；残基302 - 325）的构象重排，其中来自7个原体的环结合形成跨膜的14链β桶。为了验证该模型，我们在D2L2环的24个连续残基中产生了半胱氨酸取代，用每个突变体在人工双层膜中形成通道，并在向反侧隔室添加硫醇反应性、双层不透性试剂甲硫基磺酸乙酯三甲基铵（MTS - ET）后检查通道电导的变化。这些实验的基本原理是，MTS - ET与半胱氨酸残基反应会添加一个带正电荷的基团，因此如果该残基位于离子传导途径中，可能会降低通道电导。我们发现在两个区段（残基302 - 311和316 - 325）的连续残基中，电导交替降低和不降低。这种模式与这两个区段的交替极性和非极性残基分别伸向孔腔和双层膜一致。连接这两个区段的残基（残基312 - 315）对MTS - ET有反应，这与其处于转角区域一致。由选定突变体（H304C和N306C）形成的单通道显示出对MTS - ET的多个电导阶跃变化，这与寡聚孔一致。我们还发现通道阻断四烷基铵离子的结合位点相对于插入的D2L2环位于顺侧。这些发现构成了有力证据，支持前体孔转化为14链β桶孔的模型，并巩固了理解炭疽毒素转运机制研究的基础。
