Benabdelhak Houssain, Kiontke Stephan, Horn Carsten, Ernst Robert, Blight Mark A, Holland I Barry, Schmitt Lutz
Institut de Génétique et Microbiologie, Bât. 409, Université de Paris XI, 91405, Orsay, France.
J Mol Biol. 2003 Apr 11;327(5):1169-79. doi: 10.1016/s0022-2836(03)00204-3.
A member of the family of RTX toxins, Escherichia coli haemolysin A, is secreted from Gram-negative bacteria. It carries a C-terminal secretion signal of approximately 50 residues, targeting the protein to the secretion or translocation complex, in which the ABC-transporter HlyB is a central element. We have purified the nucleotide-binding domain of HlyB (HlyB-NBD) and a C-terminal 23kDa fragment of HlyA plus the His-tag (HlyA1), which contains the secretion sequence. Employing surface plasmon resonance, we were able to demonstrate that the HlyB-NBD and HlyA1 interact with a K(D) of approximately 4 microM. No interaction was detected between the HlyA fragment and unrelated NBDs, OpuAA, involved in import of osmoprotectants, and human TAP1-NBD, involved in the export of antigenic peptides. Moreover, a truncated version of HlyA1, lacking the secretion signal, failed to interact with the HlyB-NBD. In addition, we showed that ATP accelerated the dissociation of the HlyB-NBD/HlyA1 complex. Taking these results together, we propose a model for an early stage of initiation of secretion in vivo, in which the NBD of HlyB, specifically recognizes the C terminus of the transport substrate, HlyA, and where secretion is initiated by subsequent displacement of HlyA from HlyB by ATP.
RTX毒素家族的成员之一，大肠杆菌溶血素A，是从革兰氏阴性细菌中分泌出来的。它带有一个约50个残基的C末端分泌信号，将该蛋白靶向分泌或转运复合体，其中ABC转运蛋白HlyB是核心元件。我们已经纯化了HlyB的核苷酸结合结构域（HlyB-NBD）以及HlyA的一个C末端23kDa片段加His标签（HlyA1），HlyA1包含分泌序列。利用表面等离子体共振，我们能够证明HlyB-NBD和HlyA1以约4 microM的解离常数（K(D)）相互作用。在HlyA片段与参与渗透保护剂导入的无关NBD（OpuAA）以及参与抗原肽输出的人TAP1-NBD之间未检测到相互作用。此外，缺少分泌信号的HlyA1截短版本未能与HlyB-NBD相互作用。另外，我们表明ATP加速了HlyB-NBD/HlyA1复合体的解离。综合这些结果，我们提出了一个体内分泌起始早期阶段的模型，其中HlyB的NBD特异性识别转运底物HlyA的C末端，并且分泌是由随后ATP将HlyA从HlyB上置换而启动的。
