Structural and mechanistic insights into Helicobacter pylori NikR activation

Area of Science:

• Microbiology
• Molecular Biology
• Structural Biology

Background:

• NikR is a transcriptional metalloregulator in Helicobacter pylori, essential for responding to acidic environments.
• HpNikR controls gene expression by binding to promoter regions, a process influenced by nickel (Ni(II)) binding.
• Previous models proposed Ni(II) binding to high-affinity (HA) and external (X) sites for HpNikR activation.

Purpose of the Study:

• To biochemically and structurally characterize Helicobacter pylori NikR (HpNikR) mutants.
• To elucidate the role of metal-binding sites and protein interfaces in HpNikR DNA binding and activation.
• To develop a refined model for HpNikR-DNA interaction and transcriptional regulation.

Main Methods:

• Biochemical characterization of HpNikR mutants.
• Small-angle X-ray scattering (SAXS) to determine solution structure.
• X-ray crystallography of selected HpNikR mutants.
• Analysis of DNA-binding affinities and metallation effects.

Main Results:

• High-affinity (HA) metal-binding sites are essential but insufficient for HpNikR DNA binding.
• Secondary external (X) sites and dimer-dimer interface residues are critical for DNA binding.
• A second metal ion is required for HpNikR binding to specific promoters.
• HpNikR adopts a defined solution conformation, with nickel not inducing major structural changes.
• Crystal structures revealed the impact of mutations on HpNikR structure.

Conclusions:

• HpNikR DNA binding requires HA sites and an hydrogen bond network.
• Metallation of the unique secondary external (X) site modulates binding to low-affinity promoters by disrupting a salt bridge.
• A new model for HpNikR activation is proposed, integrating structural and biochemical data.