The N-terminal Arm of the Helicobacter pylori Ni2+-dependent Transcription Factor NikR Is Required for Specific DNA Binding

Abstract

The Ni 2؉ -dependent transcription factor NikR is widespread among microbes. The two experimentally characterized NikR orthologs, from Helicobacter pylori and Escherichia coli, display vastly different regulatory capabilities in response to increased intracellular Ni 2؉ . Here, we demonstrate that the nine-residue N-terminal arm present in H. pylori NikR plays a critical role in the expanded regulatory capabilities of this NikR family member. Specifically, the N-terminal arm is required to inhibit NikR binding to low affinity and nonspecific DNA sequences and is also linked to a cation requirement for NikR binding to the nixA promoter. Site-directed mutagenesis and arm-truncation variants of NikR indicate that two residues, Asp-7 and Asp-8, are linked to the cation requirement for binding. Pro-4 and Lys-6 are required for maximal DNA binding affinity of the full-length protein to both the nixA and ureA promoters. The N-terminal arm is highly variable among NikR family members, and these results suggest that it is an adaptable structural feature that can tune the regulatory capabilities of NikR to the nickel physiology of the microbe in which it is found.

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

“…The presence of each mutation was verified by DNA sequencing (SeqWright, Houston, TX). Protein Expression and Purification-All HpNikR proteins and mutant variants were expressed and purified as described previously (5). The T3C, N5C, and N20C mutant proteins were purified with 1 mM ␤-mercaptoethanol present throughout the purification protocol.…”

“…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.…”

“…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).…”

“…In previous work, alanine scanning and truncation mutagenesis indicated that the nine-residue N-terminal arm of HpNikR exhibits conformational differences when the protein is bound to the nixA and ureA promoters, based on changes in DNA binding affinity (5). Here, we have further examined this conformational difference using chemically modified and new sitedirected mutants of HpNikR, in combination with engineered promoter sequences, to demonstrate that protein conformational differences exist beyond the N-terminal arm when the protein is bound to the nixA and ureA promoters.…”

“…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).…”