Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur

Abstract

Zinc is one of the essential transition metals in cells. Excess or lack of zinc is detrimental, and cells exploit highly sensitive zinc-binding regulators to achieve homeostasis. In this article, we present a crystal structure of active Zur from Streptomyces coelicolor with three zinc-binding sites (C-, M-, and D-sites). Mutations of the three sites differentially affected sporulation and transcription of target genes, such that C- and M-site mutations inhibited sporulation and derepressed all target genes examined, whereas D-site mutations did not affect sporulation and derepressed only a sensitive gene. Biochemical and spectroscopic analyses of representative metal site mutants revealed that the C-site serves a structural role, whereas the M- and D-sites regulate DNA-binding activity as an on-off switch and a fine-tuner, respectively. Consistent with differential effect of mutations on target genes, zinc chelation by TPEN derepressed some genes ( znuA, rpmF2 ) more sensitively than others ( rpmG2 , SCO7682) in vivo. Similar pattern of TPEN-sensitivity was observed for Zur-DNA complexes formed on different promoters in vitro. The sensitive promoters bound Zur with lower affinity than the less sensitive ones. EDTA-treated apo-Zur gained its DNA binding activity at different concentrations of added zinc for the two promoter groups, corresponding to free zinc concentrations of 4.5 × 10 −16 M and 7.9 × 10 −16 M for the less sensitive and sensitive promoters, respectively. The graded expression of target genes is a clever outcome of subtly modulating Zur-DNA binding affinities in response to zinc availability. It enables bacteria to detect metal depletion with improved sensitivity and optimize gene-expression pattern.

Characterization of the Response to Zinc Deficiency in the Cyanobacterium Anabaena sp. Strain PCC 7120

“…Zur binding and dissociation from DNA are governed by the occupancy of a regulatory coordination site. Recent crystal structures have revealed three potential metal-coordinating sites in Zur from distinct bacteria (39,55). The role of site 3 is controversial (41,55), and it has been proposed that in vivo, zinc never binds to this site (41).…”

“…Thus, this family includes members like Zur, Fur, Nur, and Mur, which sense distinct divalent metals (Zn, Fe, Ni, and Mn, respectively), and PerR and Irr, which sense cytoplasmic peroxides and heme, respectively (33). Like other members of the Fur family, Zur proteins have two structural domains connected by a mobile hinge, an N-terminal winged-helix DNA-binding domain and a C-terminal dimerization domain (39,55). Zur contains several coordination sites for zinc (39,41,55) and senses the cytoplasmic concentration of exchangeable zinc by binding to this metal, which in turn allows Zur binding to DNA (33).…”

“…Like other members of the Fur family, Zur proteins have two structural domains connected by a mobile hinge, an N-terminal winged-helix DNA-binding domain and a C-terminal dimerization domain (39,55). Zur contains several coordination sites for zinc (39,41,55) and senses the cytoplasmic concentration of exchangeable zinc by binding to this metal, which in turn allows Zur binding to DNA (33). Effective sensing of zinc is presumed to require the concentration of the metal in the cytoplasm to approach the affinity of the regulatory coordination site(s) (41,55), and zinc has been estimated to be present in subpicomolar concentrations in Escherichia coli (45).…”

“…Zur contains several coordination sites for zinc (39,41,55) and senses the cytoplasmic concentration of exchangeable zinc by binding to this metal, which in turn allows Zur binding to DNA (33). Effective sensing of zinc is presumed to require the concentration of the metal in the cytoplasm to approach the affinity of the regulatory coordination site(s) (41,55), and zinc has been estimated to be present in subpicomolar concentrations in Escherichia coli (45). Occupancy of the regulatory site probably induces a conformational reorientation of the two domains so that, in the dimer, the DNA-binding domains adopt an optimal orientation for binding to DNA (39,41,55).…”

The Fur homologue BosR requires Arg39 to activate rpoS transcription in Borrelia burgdorferi and thereby direct spirochaete infection in mice

“…Pfam alignments of FUR proteins (PF01475) show that BosR's Arg39 aligns with a highly conserved arginine present in virtually all FUR proteins. X-ray crystallography studies show that FUR proteins consist of two domains: an N-terminal DNA-binding domain and a C-terminal dimerization domain Butcher et al, 2012;Dian et al, 2011;Gilston et al, 2014;Jacquamet et al, 2009;Lin et al, 2014;Lucarelli et al, 2007;Makthal et al, 2013;Pohl et al, 2003;Sheikh & Taylor, 2009;Shin et al, 2011;Traoré et al, 2006Traoré et al, , 2009. The DNA-binding domain contains a winged helix-turn-helix (wHTH) motif that provides the protein with the means to bind DNA (Pohl et al, 2003).…”