Institute of Microbiology of the CAS, v. v. i., Prague, Czech Republic.
Faculty of Science, Charles University, Prague, Czech Republic.
Sci Rep. 2017 Aug 24;7(1):9330. doi: 10.1038/s41598-017-09575-6.
The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) translocates its adenylate cyclase (AC) enzyme domain into target cells in a step that depends on membrane cholesterol content. We thus examined what role in toxin activities is played by the five putative cholesterol recognition amino acid consensus (CRAC) motifs predicted in CyaA hemolysin moiety. CRAC-disrupting phenylalanine substitutions had no impact on toxin activities and these were not inhibited by free cholesterol, showing that the putative CRAC motifs are not involved in cholesterol binding. However, helix-breaking proline substitutions in these segments uncovered a structural role of the Y632, Y658, Y725 and Y738 residues in AC domain delivery and pore formation by CyaA. Substitutions of Y940 of the fifth motif, conserved in the acylated domains of related RTX toxins, did not impact on fatty-acylation of CyaA by CyaC and the CyaA-Y940F mutant was intact for toxin activities on erythrocytes and myeloid cells. However, the Y940A or Y940P substitutions disrupted the capacity of CyaA to insert into artificial lipid bilayers or target cell membranes. The aromatic ring of tyrosine 940 side chain thus appears to play a key structural role in molecular interactions that initiate CyaA penetration into target membranes.
腺苷酸环化酶毒素-溶血素（CyaA、ACT 或 AC-Hly）将其腺苷酸环化酶（AC）酶结构域转运到靶细胞中，这一步骤依赖于膜胆固醇含量。因此，我们研究了在 CyaA 溶血素部分预测的五个假定的胆固醇识别氨基酸共有序列（CRAC）基序在毒素活性中起什么作用。破坏 CRAC 的苯丙氨酸取代对毒素活性没有影响，并且不受游离胆固醇抑制，表明假定的 CRAC 基序不参与胆固醇结合。然而，这些片段中的破环脯氨酸取代揭示了 Y632、Y658、Y725 和 Y738 残基在 AC 结构域递呈和 CyaA 形成孔中的结构作用。第五个基序中保守的 Y940 取代（相关 RTX 毒素酰化结构域中的保守残基）不影响 CyaA 被 CyaC 的酰化，并且 CyaA-Y940F 突变体在红细胞和髓样细胞上具有完整的毒素活性。然而，Y940A 或 Y940P 取代破坏了 CyaA 插入人工脂质双层或靶细胞膜的能力。因此，酪氨酸 940 侧链的芳环似乎在启动 CyaA 穿透靶细胞膜的分子相互作用中起着关键的结构作用。
