Sequential binding and sensing of Zn(II) by Bacillus subtilis Zur

Abstract

Bacillus subtilis Zur (BsZur) represses high-affinity zinc-uptake systems and alternative ribosomal proteins in response to zinc replete conditions. Sequence alignments and structural studies of related Fur family proteins suggest that BsZur may contain three zinc-binding sites (sites 1–3). Mutational analyses confirm the essential structural role of site 1, while mutants affected in sites 2 and 3 retain partial repressor function. Purified BsZur binds a maximum of two Zn(II) per monomer at site 1 and site 2. Site 3 residues are important for dimerization, but do not directly bind Zn(II). Analyses of metal-binding affinities reveals negative cooperativity between the two site 2 binding events in each dimer. DNA-binding studies indicate that BsZur is sequentially activated from an inactive dimer (Zur2:Zn2) to a partially active asymmetric dimer (Zur2:Zn3), and finally to the fully zinc-loaded active form (Zur2:Zn4). BsZur with a C84S mutation in site 2 forms a Zur2:Zn3 form with normal metal- and DNA-binding affinities but is impaired in formation of the Zur2:Zn4 high affinity DNA-binding state. This mutant retains partial repressor activity in vivo, thereby supporting a model in which stepwise activation by zinc serves to broaden the physiological response to a wider range of metal concentrations.