H-093. Fur Family Transcriptional Regulators in Bacillus subtilis: The Role of Three Conserved Metal Binding Motifs in Sensing Metals

S. E. Gabriel, J-W. Lee, A. R. Abberbock, J. D. Helmann;
Cornell Univ., Ithaca, NY.

B. subtilis encodes three members of the Fur family of transcriptional regulators: Fur controls iron homeostasis, Zur controls zinc homeostasis, and PerR regulates a peroxide stress response. While the proteins controlled by each these regulators have been studied extensively, the structural basis of metal ion sensing has yet to be elucidated. Published crystal structures of Fur family members have revealed the existence of as many as three distinct metal binding sites. Our biochemical and genetic analysis of these Fur family members suggests that each protein monomer contains a high affinity, Cys4-Zn(II) site important for protein folding (site 1), a metal-sensing site that binds either Fe(II) (Fur and PerR) or Zn(II) (Zur) (site 2), and a third, poorly defined site (site 3) that may allosterically modulate the affinity or specificity of the sensing site. This model differs from that proposed in the crystal structure of P. aeruginosa Fur in which site 2 was a proposed zinc site, site 3 was postulated to sense Fe(II) and no Cys4:Zn site was observed. A suite of mutants for each of the three sites in both proteins has been generated. In our published work, the roles of sites 1 and 2 in PerR have been documented: site 1 binds an essential structural Zn ion and site 2 senses peroxides through metal catalyzed protein oxidation. Here, we demonstrate that mutations in PerR site 3 function as a repressor in the presence of Mn(II), but not Fe(II). The precise role of site 3 in modulating site 2 function is not yet clear. To determine whether or not the functions of these three sites also applies to other Fur paralogs, we have generated a set of site-directed mutants in Zur. As expected based on the results with PerR, mutations in site 1 abolish Zur function (by destabilizing the protein) and mutants in site 2 mutants show a reduced ability to sense zinc. Further, mutations in the Zur site 3 (which is located adjacent to site 2) affect site 2 activity. In summary, our work has determined a function for site 1 and site 2 for this family of regulators and our current hypothesis is that site 3 acts as a secondary regulatory site which modulates the ability for metal to be bound at site 2.

238/H. DNA Repair, Recombination and Replication

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