The structure of the Helicobacter pylori ferric uptake regulator Fur reveals three functional metal binding sites

Abstract

SummaryFur, the ferric uptake regulator, is a transcription factor that controls iron metabolism in bacteria. Binding of ferrous iron to Fur triggers a conformational change that activates the protein for binding to specific DNA sequences named Fur boxes. In Helicobacter pylori, HpFur is involved in acid response and is important for gastric colonization in model animals. Here we present the crystal structure of a functionally active HpFur mutant (HpFur2M; C78S-C150S) bound to zinc. Although its fold is similar to that of other Fur and Fur-like proteins, the crystal structure of HpFur reveals a unique structured N-terminal extension and an unusual C-terminal helix. The structure also shows three metal binding sites: S1 the structural ZnS 4 site previously characterized biochemically in HpFur and the two zinc sites identified in other Fur proteins. Site-directed mutagenesis and spectroscopy analyses of purified wild-type HpFur and various mutants show that the two metal binding sites common to other Fur proteins can be also metallated by cobalt. DNA protection and circular dichroism experiments demonstrate that, while these two sites influence the affinity of HpFur for DNA, only one is absolutely required for DNA binding and could be responsible for the conformational changes of Fur upon metal binding while the other is a secondary site.

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

“…Because D-sites are occupied by zinc and M-sites can accommodate iron, the D-site in these proteins is regarded to serve a structural role. On the other hand, a recent report on the structure of active zinc-bound variant of HpFur, which contains C-terminal cysteines, revealed the occupation of the D-site in addition to M-and C-sites (29). In this structure, the D-site metallation has been proposed to be nonessential for DNAbinding, but to serve a secondary role to modulate binding affinity, consistent with what we discovered in the present study.…”

Ferric Uptake Regulator and Its Role in the Pathogenesis of Nontypeable Haemophilus influenzae

“…The Fur crystal structures from these microorganisms have been solved and contain a series of ␣-helices and ␤-sheets characteristic of a typical, DNAbinding, winged helix-turn-helix motif. In addition, Fur from V. cholerae, P. aeruginosa, and H. pylori have an N-terminal DNAbinding domain and a C-terminal dimerization domain (45)(46)(47)(48). The Fur protein from NTHi strain 86-028NP contains residues (D103 and R109) predicted to be important for both Fur dimerization and DNA-binding affinity (46) and a tyrosine (Y55) predicted to be essential for DNA binding (45).…”

“…The Fur protein from NTHi strain 86-028NP contains residues (D103 and R109) predicted to be important for both Fur dimerization and DNA-binding affinity (46) and a tyrosine (Y55) predicted to be essential for DNA binding (45). The Fur protein from NTHi strain 86-028NP also contains four cysteines in the dimerization domain predicted to be involved in zinc coordination (45,47). Finally, the Fur protein from NTHi strain 86-028NP contains an HHDH motif in the region where the DNA-binding domain overlaps with the dimerization domain and two glutamic acids and a histidine within the dimerization domains.…”

Fur-Mediated Global Regulatory Circuits in Pathogenic Neisseria Species

“…In most organisms, Fur is present as a 15-to-17-kDa protein that forms dimers in the presence of iron (II) or other divalent cations (6,24,74,91). As revealed by analysis of several crystal structures of the Fur protein from various pathogens, the amino terminus of Fur has been shown to bind to DNA whereas the carboxyl terminus is involved in dimer formation (26,81,91). Analysis of the crystal structure of Fur has also identified multiple metal-binding sites.…”