Department of Microbiology and Molecular Genetics, The University of Texas - Houston Health Science Center, Medical School, Houston, TX, USA.
Mol Microbiol. 2011 Nov;82(3):634-47. doi: 10.1111/j.1365-2958.2011.07843.x. Epub 2011 Oct 10.
AtxA, a unique regulatory protein of unknown molecular function, positively controls expression of the major virulence genes of Bacillus anthracis. The 475 amino acid sequence of AtxA reveals DNA binding motifs and regions similar to proteins associated with the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). We used strains producing native and functional epitope-tagged AtxA proteins to examine protein-protein interactions in cell lysates and in solutions of purified protein. Co-affinity purification, non-denaturing polyacrylamide gel electrophoresis and bis(maleimido)hexane (BMH) cross-linking experiments revealed AtxA homo-multimers. Dimers were the most abundant species. BMH cross-links available cysteines within 13 Å. To localize interaction sites, six AtxA mutants containing distinct Cys→Ser substitutions were tested for multimerization and cross-linking. All mutants multimerized, but one mutation, C402S, prevented cross-linking. Thus, BMH uses C402 to make the inter-molecular bond between AtxA proteins, but C402 is not required for protein-protein interaction. C402 is in a region bearing amino acid similarity to Enzyme IIB proteins of the PTS. The AtxA EIIB motif may function in protein oligomerization. Finally, cultures grown with elevated CO(2) /bicarbonate exhibited increased AtxA dimer/monomer ratios and increased AtxA activity, relative to cultures grown without added CO(2) /bicarbonate, suggesting that this host-associated signal enhances AtxA function by shifting the dimer/monomer equilibrium towards the dimeric state.
AtAx 是一种未知分子功能的独特调控蛋白，正向调控炭疽杆菌主要毒力基因的表达。AtAx 的 475 个氨基酸序列揭示了 DNA 结合基序和与磷酸烯醇丙酮酸：碳水化合物磷酸转移酶系统 (PTS) 相关的蛋白相似的区域。我们使用产生天然和功能表位标记 AtxA 蛋白的菌株，在细胞裂解物和纯化蛋白溶液中检查蛋白-蛋白相互作用。共亲和纯化、非变性聚丙烯酰胺凝胶电泳和双（马来酰亚胺）己烷 (BMH) 交联实验揭示了 AtxA 同源多聚体。二聚体是最丰富的物种。BMH 交联可利用 13Å 内的半胱氨酸。为了定位相互作用位点，测试了六个含有不同 Cys→Ser 取代的 AtxA 突变体的多聚化和交联。所有突变体都发生了多聚化，但一个突变体 C402S 阻止了交联。因此，BMH 使用 C402 在 AtxA 蛋白之间形成分子间键，但 C402 不是蛋白-蛋白相互作用所必需的。C402 位于与 PTS 的 Enzyme IIB 蛋白具有氨基酸相似性的区域。AtAx 的 EIIB 基序可能在蛋白寡聚化中发挥作用。最后，与不添加 CO(2) /碳酸氢盐的培养物相比，在升高的 CO(2) /碳酸氢盐中生长的培养物表现出更高的 AtxA 二聚体/单体比和更高的 AtxA 活性，表明这种宿主相关的信号通过将二聚体/单体平衡向二聚体状态转移来增强 AtxA 功能。
