title: 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 (Zur(2):Zn(2)) to a partially active asymmetric dimer (Zur(2):Zn(3)), and finally to the fully zinc-loaded active form (Zur(2):Zn(4)). BsZur with a C84S mutation in site 2 forms a Zur(2):Zn(3) form with normal metal- and DNA-binding affinities but is impaired in formation of the Zur(2):Zn(4) 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.
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Ma
authors.forename: Scott E
authors.initials: SE
authors.lastname: Gabriel
authors.forename: John D
authors.initials: JD
authors.lastname: Helmann
mesh_terms: D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D009154:Mutation; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; D004247:DNA; D015032:Zinc