Helicobacter pylori NikR Protein Exhibits Distinct Conformations When Bound to Different Promoters

Abstract

Helicobacter pylori requires nickel ions as cofactors for the urease and hydrogenase enzymes, which are important for the colonization of animal models of infection (1-3). This virulence requirement has motivated numerous studies of nickel-dependent gene regulation in H. pylori. The Ni 2ϩ -responsive transcription factor NikR from H. pylori (HpNikR) 3 regulates the expression of multiple genes by directly binding to their promoters in response to increasing nickel (4 -10). HpNikR is an exquisitely sensitive nickel sensor with a K D,Ni of ϳ2 pM (4, 5); however, how that affinity translates to gene regulation in cells remains unclear (e.g. in rich media, changes in gene expression are observed in response to ϳ100 M) (6, 9 -11). From these studies, it appears that the recognition sequences in each promoter are defined by a series of poorly conserved 6-bp imperfect inverted repeat half-sites separated by a 15-bp spacer (5,7,12). HpNikR binds to these promoters with a range of affinities (5, 12). The promiscuity of DNA binding by HpNikR contrasts with high sequence selectivity of the NikR proteins from Escherichia coli (EcNikR (13, 14)) and Geobacter uraniireducens (GuNikR (15)), which recognize highly conserved inverted repeats found in only one or two promoter regions per genome. The NikR protein family represents an excellent system with which to understand the role of protein and DNA sequence in the expansion of prokaryotic regulatory networks.NikR proteins are ribbon-helix-helix (RHH) transcription factors. RHH family members are present in bacteria, archaea, and bacteriophages (16). These proteins display diverse regulatory functions, such as bacteriophage gene regulation (17), plasmid maintenance and segregation (18,19), plasmid antitoxin and repressor functions (20), and metabolite-dependent (21) and metal-dependent (13, 22) gene regulation.The archetypical RHH fold (ϳ45-50 amino acids) consists of a single ␤-strand followed by two ␣-helices, with the two ␤-strands of each obligate RHH dimer forming an antiparallel ␤-sheet motif (23). Structural studies of the Arc and MetJ repressors first demonstrated that the RHH ␤-sheet motif sits in the major groove of DNA, with three solvent-exposed residues making base-specific hydrogen bonds via their side chains (23)(24)(25). Nonspecific DNA phosphate contacts were also observed in the structures, some by tandem turn regions N-terminal to the ␤-sheets and others by the N terminus of helix ␣2 of the RHH domain (23-25). These protein-phosphate interactions are hypothesized to attach the N terminus and helix ␣2 to the DNA backbone and link these structural elements to the ␤-sheet (23). The helix ␣2-phosphate backbone interactions are shared among all of the RHH protein-DNA co-crystal structures (16, 24 -30

On the interaction of Helicobacter pylori NikR, a Ni(II)-responsive transcription factor, with the urease operator: in solution and in silico studies

“…Therefore, even though a tighter affinity to OP ureA has been reported for the form of HpNikR containing a mixed Ni(II) coordination as compared to the regulator form that binds Ni(II) only in square planar sites [23], the real impact of this mixed form could be established only when a crystal structure of the mixed coordinated HpNikR-DNA complex will be made available. Conflicting data have also been reported for the role of other metal-binding sites, in addition to the HHHC sites, having an effect on the binding constant between HpNikR and different DNA sequences [25,29,53,[62][63][64][65][66][67].…”

Nickel-responsive regulation of two novelHelicobacter pyloriNikR-targeted genes

“…However, several HpNikRcontrolled genes have been identified that lack any sequence homology with the proposed consensus, 22 consistent with a more complicated strategy for DNA recognition by HpNikR. 30 Understanding the complete physiological function of HpNikR requires that all of the HpNikR targets be identified. To this end, we performed a chromatin immunoprecipitation experiment followed by sequencing (ChIP-seq), which revealed the promoters of HPG27_866 and HPG27_1499.…”

Metallochaperones and metalloregulation in bacteria

“…Non-cognate metal ions Zn II and Co II bind with different affinities, stoichiometries and form non-native coordination geometries and ligand sets that do not support a metal-mediated allosteric response [160,166]. Ni II also binds to lower binding affinity Ni II sites leading to an enhancement of DNA binding affinity [166,167], while different conformations have been shown to bind different promoters [168–170]. NikR thus provides an exceptional illustration of the importance of metal coordination geometry as a key feature of cognate metal sensing by a metalloregulatory protein, that is reinforced by the prevailing intracellular metalmilieu.…”