Bacillus anthracis virulence regulator AtxA: oligomeric state, function and CO2-signalling
Bacillus anthracis virulence regulator AtxA: oligomeric state, function and CO2-signalling
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DOI：
10.1111/j.1365-2958.2011.07843.x
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发表时间：
2011-11-01
影响因子：
3.6
通讯作者：
Koehler, Theresa M.
中科院分区：
文献类型：
--
作者：
Hammerstrom, Troy G.;Roh, Jung Hyeob;Koehler, Theresa M.
AtxA是一种分子功能未知的独特调控蛋白，正向控制炭疽芽孢杆菌主要毒力基因的表达。Atxa的475个氨基酸序列显示了与磷酸烯醇式丙酮酸：碳水化合物磷酸转移酶系统(PTS)相关的蛋白质的DNA结合基序和区域。我们使用产生天然和功能性表位标记的Atxa蛋白的菌株来检查细胞裂解产物和纯化蛋白溶液中的蛋白质相互作用。共亲和纯化、非变性聚丙烯酰胺凝胶电泳法和双(马来酰亚胺)正己烷(BMH)交联实验表明ATXA是多聚体。二聚体是最丰富的物种。BMH交联可用半胱氨酸在13埃内。为了定位相互作用位点，我们测试了6个含有不同Cys->；Ser取代的Atxa突变体的多聚化和交联性。所有的突变体都是多聚体，但有一个突变C402S阻止了交叉连接。因此，BMH使用C402在Atxa蛋白之间建立分子间键，但C402不是蛋白质相互作用所必需的。C402位于与PTS的IIB酶蛋白氨基酸相似的区域。Atxa EIIB基序可能在蛋白质齐聚中发挥作用。最后，与没有添加二氧化碳/重碳酸盐的培养物相比，高CO2/重碳酸盐培养的培养物表现出更高的atxa二聚体/单体比和更高的atxa活性，这表明这种与宿主相关的信号通过使二聚体/单体平衡向二聚体状态移动而增强了atxa的功能。
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 angstrom. 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 CO2/bicarbonate exhibited increased AtxA dimer/monomer ratios and increased AtxA activity, relative to cultures grown without added CO2/bicarbonate, suggesting that this host-associated signal enhances AtxA function by shifting the dimer/monomer equilibrium towards the dimeric state.