Molecular basis for the integration of environmental signals by FurB from Anabaena sp. PCC 7120

Abstract

FUR (Ferric uptake regulator) proteins are among the most important families of transcriptional regulators in prokaryotes, often behaving as global regulators. In the cyanobacterium PCC 7120, FurB (Zur, Zinc uptake regulator) controls zinc and redox homeostasis through the repression of target genes in a zinc-dependent manner., non-specific binding of FurB to DNA elicits protection against oxidative damage and avoids cleavage by deoxyribonuclease I. The present study provides, for the first time, evidence of the influence of redox environment in the interaction of FurB with regulatory zinc and its consequences in FurB-DNA-binding affinity. Calorimetry studies showed that, in addition to one structural Zn(II), FurB is able to bind two additional Zn(II) per monomer and demonstrated the implication of cysteine C93 in regulatory Zn(II) coordination. The interaction of FurB with the second regulatory zinc occurred only under reducing conditions. While non-specific FurB-DNA interaction is Zn(II)-independent, the optimal binding of FurB to target promoters required loading of two regulatory zinc ions. Those results combined with site-directed mutagenesis and gel-shift assays evidenced that the redox state of cysteine C93 conditions the binding of the second regulatory Zn(II) and, in turn, modulates the affinity for a specific DNA target. Furthermore, differential spectroscopy studies showed that cysteine C93 could also be involved in heme coordination by FurB, either as a direct ligand or being located near the binding site. The results indicate that besides controlling zinc homeostasis, FurB could work as a redox-sensing protein probably modifying its zinc and DNA-binding abilities depending upon environmental conditions.

Bacterial zinc uptake regulator proteins and their regulons

“…Cell lysis in presence of 2 M GdnHCl (pH 8). Zn-IMAC, dialysis pH 5.5 Mostly monomer, some dimer (SDS–PAGE, denaturing conditions) 1 (ICP-MS) Up to two more binding sites per monomer [ 48 ] Abbreviations: AEX: anion-exchange chromatography; DTT: dithiothreitol; EDTA: ethylenediaminetetraacetic acid; FAAS: flame atomic absorption spectroscopy; GdnHCl: guanidinium hydrochloride; GST: Glutathione S -transferase; HIC: hydrophobic interaction chromatography; His-tag: polyhistidine-tag; ICP-AES/ICP-OES: inductively coupled plasma atomic/optical emission spectroscopy; ICP-MS: inductively coupled plasma mass spectrometry; IMAC: immobilised metal ion affinity chromatography; MBP: maltose-binding protein; n.d: not determined; μPIXE: micro-proton-induced X-ray emission; PAR: pyridyl-azo-resorcinol; SDS–PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SEC: size-exclusion chromatography; TEV: tobacco etch virus; WT: wild type. …”

“…Furthermore, the pleiotropic phenotype of Δ zur and zur -overexpressing mutants, extensive cross-talk between Zur, Fur, and PerR, and up-regulation of antioxidant enzymes (superoxide dismutase sodA, catalase alr0998, and peroxiredoxin gct3) in a Δ zur mutant of the cyanobacterium Anabaena sp. PCC 7120 probably also include both direct and indirect effects [ 48 , 62 , 97 , 98 ]. In turn, the levels of metal ions other than Zn 2+ may also affect the expression of members of Zur regulons, as shown for Enterococcus faecalis [ 76 ], where exposure to high levels of Cu 2+ activates Zur-regulated zinc uptake, presumably to protect from mis-metallation of Zn-requiring proteins and from oxidative stress.…”

A single sensor controls large variations in zinc quotas in a marine cyanobacterium

“…Some biochemical and/or functional studies on the three homologs from freshwater Anabaena spp. PCC 7120 are available 19 , 21 . However, owing to the absence of structural data for any cyanobacterial Fur-family protein, no clear structure–function relationship has been established, the residues imparting metal specificity are unknown, and hence these annotations have remained tentative.…”

Metal binding and oligomerization properties of FurC (PerR) from Anabaena sp. PCC7120: an additional layer of regulation?

“…PCC7120 (herein Anabaena sp.) lead to different ways of interaction of these proteins with DNA [9,10].…”