Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin
Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin
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DOI：
10.1074/jbc.m803334200
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发表时间：
2008-08-29
影响因子：
4.8
通讯作者：
Satchell, Karla J. Fullner
中科院分区：
文献类型：
--
作者：
Prochazkova, Katerina;Satchell, Karla J. Fullner
霍乱弧菌分泌一种与毒力相关的多功能自动处理 RTX 毒素 (MARTXVc)。通过嵌入的半胱氨酸蛋白酶结构域 (CPD) 对这种毒素进行自动处理对于这种毒素在多种细胞类型中诱导肌动蛋白解聚至关重要。大型梭菌毒素 TcdB 中也存在同源 CPD，最近的研究表明，六磷酸肌醇（Ins(1,2,3,4,5,6) P6 或 InsP6）刺激 TcdB 依赖于 CPD 的自动加工（Egerer, M.、Giesemann, T.、Jank, T.、Satchell, K. J. 和 Aktories, K. (2007)化学杂志，282，25314-25321）。在这项工作中，MARTXVc 内 CPD 的自动处理活动同样被发现是由 InsP6 诱导的。 CPD 显示可结合 InsP6（Kd，0.6 μM），并且 InsP6 在生理浓度和低至 0.01 μM 浓度下均能刺激分子内自动加工。处理后的 CPD 不结合 InsP6，表明在裂解后，活化的 CPD 可能会转变为非活性构象。为了进一步探究自动加工机制，对 24 个已鉴定的 CPD 中的保守残基进行了诱变。除了形成催化位点的半胱氨酸和组氨酸残基外，还鉴定出了 InsP6 结合所必需的 2 个赖氨酸残基以及导致低 InsP6 浓度下活性丧失的 5 个赖氨酸和精氨酸残基。总体而言，我们的数据支持这样一个模型：位于 CPD 结构上的碱性残基形成 InsP6 结合口袋，并且 InsP6 的结合通过将 CPD 改变为激活构象来刺激加工。处理后，InsP6 显示被回收，而裂解的 CPD 无法进一步结合 InsP6。
Vibrio cholerae secretes a large virulence-associated multifunctional autoprocessing RTX toxin ( MARTXVc). Autoprocessing of this toxin by an embedded cysteine protease domain (CPD) is essential for this toxin to induce actin depolymerization in a broad range of cell types. A homologous CPD is also present in the large clostridial toxin TcdB and recent studies showed that inositol hexakisphosphate (Ins(1,2,3,4,5,6) P6 or InsP6) stimulated the autoprocessing of TcdB dependent upon the CPD ( Egerer, M., Giesemann, T., Jank, T., Satchell, K. J., and Aktories, K. (2007) J. Biol. Chem. 282, 25314-25321). In this work, the autoprocessing activity of the CPD within MARTXVc is similarly found to be inducible by InsP6. The CPD is shown to bind InsP6 ( Kd, 0.6 mu M), and InsP6 is shown to stimulate intramolecular autoprocessing at both physiological concentrations and as low as 0.01 mu M. Processed CPD did not bind InsP6 indicating that, subsequent to cleavage, the activated CPD may shift to an inactive conformation. To further pursue the mechanism of autoprocessing, conserved residues among 24 identified CPDs were mutagenized. In addition to cysteine and histidine residues that form the catalytic site, 2 lysine residues essential for InsP6 binding and 5 lysine and arginine residues resulting in loss of activity at low InsP6 concentrations were identified. Overall, our data support a model in which basic residues located across the CPD structure form an InsP6 binding pocket and that the binding of InsP6 stimulates processing by altering the CPD to an activated conformation. After processing, InsP6 is shown to be recycled, while the cleaved CPD becomes incapable of further binding of InsP6.