Function and structure of the novel haem-binding protein HbpS from streptomycetes

新发现的HbpS-SenS-SenR三组分系统保护网状链霉菌免受氧化应激相关事件的影响，并在链霉菌和其他放线菌的基因组中编码。HbpS是一种新型的铁和血红素结合蛋白，组装成八聚体，并与传感器激酶SenS相互作用。基于生理学和遗传学研究，阐明的三维晶体结构以及蛋白质动力学（FRET和EPR），我们发现，在Fe 2 +/H2 O2为基础的氧化应激下，HbpS蛋白发生结构变化。随后的级联反应引发了SenS的自磷酸化，进而磷酸化反应调节剂SenR，SenR控制编码过氧化氢酶-过氧化物酶CpeB的操纵子的转录，该过氧化氢酶-过氧化物酶CpeB通过分解H2 O2保护细菌免受氧化应激。基于突变研究和HbpS的3D结构，确定了三个铁结合基序D/EXXE：两个定位于八聚体的表面，一个位于八聚体的核心。这些基序对于大量Fe 2+离子（~100/八聚体）的积累是必要的，这些Fe 2+离子随后被氧化为Fe 3+。基于我们最近获得的数据，我们将改进条件以在不存在或存在我们鉴定为特异性相互作用配体的血红素B型的情况下获得野生型和突变型HbpS蛋白的晶体。所获得的3D晶体结构的比较将鉴定在HbpS八聚体中配位血红素B时的氨基酸残基以及它们的局部重排。我们已经表明，HbpS不仅结合血红素，但，也降解it. In探索潜在的催化机制，我们计划纯化所产生的血红素转化产物的同质性，并阐明其结构特征。这些研究将通过对具有改变的催化活性的设计突变蛋白的研究来补充。值得注意的是，HbpS的主要部分对应于未知功能的蛋白质结构域（命名为DUF 336），其存在于来自细菌（4803 x）、酵母（67 x）或真核生物（199 x）的许多蛋白质中。因此，我们的研究结果将具有重要意义。

The newly discovered HbpS-SenS-SenR three-component system protects Streptomyces reticuli from oxidative stress-related events, and is encoded within genomes of streptomycetes and other actinobacteria. HbpS is a novel type of iron- and haem-binding protein, assembles to an octamer, and interacts with the sensor kinase SenS. Based on physiological and genetic studies, the elucidation of the 3D crystal structure as well as protein-dynamics (FRET and EPR), we revealed that under Fe2+/H2O2-based oxidative stress, the HbpS protein undergoes structural changes. The subsequent cascade provokes the autophosphorylation of SenS that in turn phosphorylates the response regulator SenR, which governs the transcription of an operon encoding the catalase-peroxidase CpeB that protects the bacterium from oxidative stress by decomposing H2O2. Based on mutational studies and on the 3D structure of HbpS, three iron-binding motifs D/EXXE were identified: two localize on the surface and one in the core of the octamer. These motifs are necessary for the accumulation of high quantities of Fe2+ ions (~100/octamer) that are subsequently oxidized to Fe3+. Based on our recently obtained data, we shall refine conditions to obtain crystals of the wild-type and mutant HbpS proteins in the absence or presence of haem type B that we identified as the specific interacting ligand. The comparison of the gained 3D crystal structures will identify amino acid residues as well as their local rearrangements while coordinating haem B in the HbpS octamer. We have shown that HbpS not only binds haem, but, also degrades it. In order to explore the underlying catalytic mechanism, we plan to purify the generated haem-conversion products to homogeneity, and to elucidate their structural characteristics. These studies will be complemented by investigations of designed mutant proteins with altered catalytic activity. Remarkably, the major portion of HbpS corresponds to a protein domain of an unknown function (named DUF336) that occurs in numerous proteins from bacteria (4803x), Archaea (67x) or eukaryotes (199x). Thus, the results of our studies will have important implications.