NikR–operator complex structure and the mechanism of repressor activation by metal ions

Abstract

Metal ion homeostasis is critical to the survival of all cells. Regulation of nickel concentrations in Escherichia coli is mediated by the NikR repressor via nickel-induced transcriptional repression of the nickel ABC-type transporter, NikABCDE. Here, we report two crystal structures of nickel-activated E. coli NikR, the isolated repressor at 2.1 Å resolution and in a complex with its operator DNA sequence from the nik promoter at 3.1 Å resolution. Along with the previously published structure of apo-NikR, these structures allow us to evaluate functional proposals for how metal ions activate NikR, delineate the drastic conformational changes required for operator recognition, and describe the formation of a second metal-binding site in the presence of DNA. They also provide a rare set of structural views of a ligand-responsive transcription factor in the unbound, ligand-induced, and DNA-bound states, establishing a model system for the study of ligand-mediated effects on transcription factor function.crystallography ͉ DNA complex ͉ nickel ͉ transcription factor ͉ metalloregulator M etal ions are essential nutrients for all cells, but their intracellular concentrations and distribution must be tightly regulated to avoid toxicity. Nickel ions are particularly important to the physiology of microorganisms such as the model prokaryote Escherichia coli and the human gastric pathogen Helicobacter pylori. In these organisms, incorporation of nickel into enzymes such as [NiFe]-hydrogenase and urease is necessary for metabolic adaptation to changing environmental conditions (1). In E. coli, nickel is acquired via an ABC-type membrane transporter, NikABCDE (2). Transcription of the operon encoding this nickel importer, nikABCDE, is repressed in the presence of nickel by the transcription factor NikR (3, 4). NikR therefore serves as a cytoplasmic nickel sensor, stopping production of the nickel importer when intracellular levels of nickel are sufficient. In H. pylori, which requires the nickel enzyme urease to survive and colonize the acidic gastric mucus of the human stomach, NikR plays a more complex regulatory role. Transcriptome analysis of a deletion mutant and in vitro promoter-binding assays have revealed that H. pylori NikR (HpNikR) regulates transcription of not only the nickel importer and urease enzyme, but also other regulatory networks, some through repression of the gene encoding the ferric uptake regulator (Fur) protein (5-8).The NikR protein that has been characterized most extensively at the molecular level is the homolog from E. coli. E. coli NikR (EcNikR or NikR) is a homotetramer that binds one nickel(II) ion per subunit with picomolar affinity (9, 10). Stoichiometric nickel ions activate NikR to bind a 28-bp palindromic operator sequence (GTATGA-N 16 -TCATAC) within the promoter of nikABCDE with low-nanomolar affinity (9, 11). Recently, it was demonstrated that NikR can bind a variety of divalent transition metal ions with high affinity and that stoichiometric amounts of these other ions can activate N...

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

“…The greater spacing between half-sites is a consequence of the C-terminal nickel-binding domain, which is physically interposed between the RHH domains in the DNA complex. The DNA duplexes of many of the RHH-DNA cocrystal structures are bent to varying degrees (Arc, 50°(25); MetJ, 50°(24); CopG, 60° (29); FitA, 44°(52); EcNikR, 22° (27); ParR, 46°(53)), which further supports the idea that RHH proteins with different ␤-sheet motifs require variations in flanking DNA structure for optimal DNA binding. It is likely, then, that models of RHH protein-DNA interactions need to be expanded to include the analysis of DNA structural specificity outside of the minimal binding sites often predicted from lower resolution experiments.…”

“…2e, gray boxes), consistent with the location of Asn 20 at the end of helix ␣1 and near the two edges of the antiparallel ␤-sheet (Fig. 1b) (27). Because HpNikR binds to DNA as a tetramer, the presence of more than four cleavage regions suggests that each modified Cys adopted more than one conformation.…”

“…The EcNikR-operator structure revealed DNA interactions similar to those of the Arc and MetJ repressors (27). Additionally, the nickel binding C-terminal domain interacts with the phosphate backbone of the central base pairs that separate the inverted repeat sequences.…”

Structure, Function, and Targets of the Transcriptional Regulator SvtR from the Hyperthermophilic Archaeal Virus SIRV1

“…We compared the residues highlighted by the RI analysis with the residues that contact DNA or/and make dimer-dimer contacts in the available crystal structures of RHH proteins in complex with DNA, i.e. CopG, Arc, Omega, FitA, MetJ, and NikR (52,53,58,59,67,68), we identified the DNA contacting or dimerdimer contacting residues and color-coded the structurally equivalent residues of SvtR accordingly (supplemental Fig. S7).…”

The hierarchic network of metal-response transcription factors in Escherichia coli

“…NikR, the representative regulator of NikR-type transcription factors, consists of the N-terminal RHH domain and the C-terminal Ni 2+ -binding domain. [58][59][60][61] uptake at low concentrations of Zn 2+ .…”