Structural basis of low-affinity nickel binding to the nickel-responsive transcription factor NikR from Escherichia coli

Abstract

Escherichia coli NikR regulates cellular nickel uptake by binding to the nik operon in the presence of nickel and blocking transcription of genes encoding the nickel uptake transporter. NikR has two binding affinities for the nik operon: a nanomolar dissociation constant with stoichiometric nickel and a picomolar dissociation constant with excess nickel [Bloom, S. L., and Zamble, D. B. (2004) Biochemistry 43, 10029-10038; Chivers, P. T., and Sauer, R. T. (2002) Chem. Biol. 9, 1141-1148]. While it is known that the stoichiometric nickel ions bind at the NikR tetrameric interface [Schreiter, E. R., et al. (2003) Nat. Struct. Biol. 10, 794-799; Schreiter, E. R., et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 13676-13681], the binding sites for excess nickel ions have not been fully described. Here we have determined the crystal structure of NikR in the presence of excess nickel to 2.6 A resolution and have obtained nickel anomalous data (1.4845 A) in the presence of excess nickel for both NikR alone and NikR cocrystallized with a 30-nucleotide piece of double-stranded DNA containing the nik operon. These anomalous data show that excess nickel ions do not bind to a single location on NikR but instead reveal a total of 22 possible low-affinity nickel sites on the NikR tetramer. These sites, for which there are six different types, are all on the surface of NikR, and most are found in both the NikR alone and NikR-DNA structures. Using a combination of crystallographic data and molecular dynamics simulations, the nickel sites can be described as preferring octahedral geometry, utilizing one to three protein ligands (typically histidine) and at least two water molecules.

Figure 1

Overall topology of NikR with the metal binding domains (MBD) and ribbon−helix−helix (RHH) domains indicated. Representative structures of the potassium (left panel) and high-affinity nickel sites (right panel) with coordinating amino acids labeled.

Figure 2

Nickel anomalous density maps of excess nickel-soaked NikR and the NikR−DNA complex. (a) Dimer found in the asymmetric unit of the NikR structure with excess nickel ions. (b) NikR−DNA complex structure with excess nickel ions. Nickel anomalous density is shown in blue mesh at 3.5σ. Excess nickel ions are shown as cyan spheres, high-affinity nickel ions as green spheres, and potassium ions as purple spheres. Each monomer chain is colored uniquely. Nickel sites are numbered corresponding to the site types in Table 2.

Figure 3

Low-affinity nickel sites 1−3, 5, and 6 from the crystal structure of NikR without DNA. The 2F_o − F_c electron density maps at 1.0σ are colored gray around the coordinating protein ligands, water molecules, and metal ions. Nickel anomalous density maps at 3.5σ are colored blue. The coloring is the same as in Figure 2. The low-affinity nickel sites are numbered and correspond to the sites numbered in the NikR model (middle right) as well as the sites in Figure 2 and Table 2.

Figure 4

Representative structures following 1 ns molecular dynamics simulation of three types of low-affinity nickel binding sites. Simulations resulted in two types of ligand arrangements for site 2. Site types correspond to numbers in Figures 2 and 3 and Table 2.

Figure 5

Sequence alignment of NikR from E. coli (EcNikR) and H. pylori (HpNikR). High-affinity nickel ligands are highlighted in green, potassium ligands in purple, and low-affinity nickel site ligands from crystal structures in cyan, and the ligand added to site type 2 during molecular dynamics simulations (D80) is colored yellow (refer to Figure 4).

Figure 6

Proposed scheme for NikR binding DNA considering all three types of metal binding sites. Nickel ions (black circles), potassium ions (gray circles), DNA (ladder), RHH domains (triangles), ordered α3 helices (white ovals), and the central MBD (rectangle) are illustrated. As structural data indicate that excess nickel ions can bind to the protein before and after DNA binding, two pathways are depicted.