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

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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.