Structural basis of the metal specificity for nickel regulatory protein NikR

Abstract

In the presence of excess nickel, Escherichia coli NikR regulates cellular nickel uptake by suppressing the transcription of the nik operon, which encodes the nickel uptake transporter, NikABCDE. Previously published in vitro studies have shown that NikR is capable of binding a range of divalent transition metal ions in addition to Ni2+, including Co2+, Cu2+, Zn2+, and Cd2+. To understand how the high-affinity nickel binding site of NikR is able to accommodate these other metal ions, and to improve our understanding of NikR's mechanism of binding to DNA, we have determined structures of the metal-binding domain (MBD) of NikR in the apo form and in complex with Cu2+ and Zn2+ ions and compared them with the previously published structures with Ni2+. We observe that Cu2+ ions bind in a manner very similar to that of Ni2+, with a square planar geometry but with longer bond lengths. Crystals grown in the presence of Zn2+ reveal a protein structure similar to that of apo MBD with a disordered alpha3 helix, but with two electron density peaks near the Ni2+ binding site corresponding to two Zn2+ ions. These structural findings along with biochemical data on NikR support a hypothesis that ordering of the alpha3 helix is important for repressor activation.

Figure 1

Structures of E. coli NikR. Each monomer composing the tetramer is a different color. (a) Alignment of apo-MBD (in color, this work) and apo full-length NikR (in grey, PDB 1Q5V). (b) Alignment of Ni²⁺-bound structures of MBD (in color, PDB 1Q5Y) and full-length NikR (in grey, PDB 2HZA). (c) DNA- and Ni²⁺-bound full-length NikR (PDB 2HZV). K⁺, which was observed bound in NikR-DNA structure, are represented by black spheres. Asterisks indicate loops and α3 helices that make connections to the DNA.

Figure 2

Metal binding sites with 2F_o-F_c electron density (blue) contoured at 1σ around metal and ligands. One monomer is in magenta and the other in green. (a) Ni²⁺-MBD, (b) Cu²⁺-MBD, (c) Zn²⁺-MBD. The dispersive difference map (orange) was calculated by subtracting data collected at 1.2829 Å (zinc inflection wavelength) from 1.00 Å (a remote wavelength) and indicates the presence of zinc, contoured at −8.0 σ. (d) Zn²⁺-MBD with F_o-F_c difference density maps around the zinc binding sites with positive difference electron density in green contoured at +3σ and negative difference electron density in red contoured at −3σ.

Figure 3

Hydrogen bonding network connecting two nickel binding sites. Zn²⁺-MBD colored in cyan and blue aligned to the Ni²⁺-MBD structure with carbon atoms in green and magenta, oxygen in red, sulfur in yellow, and nitrogen in blue.

Figure 4

Ordering of the α3 helix. Ribbon diagrams of the (a) Ni²⁺-MBD, (b) Cu²⁺-MBD, (c) Zn²⁺-MBD, (d) apo-MBD; with each monomer represented in a separate color. (e) Circular dichroism spectra of apo-, Ni²⁺-, Cu²⁺-, and Zn²⁺-MBD.

Figure 5

Proposed NikR regulatory mechanism. Dimeric RHH domains are represented by white triangles, each monomer of the MBD is represented in a different color, bold empty rectangles represent an ordered α3 helix, nickel ions are represented by green circles outlined in black, and potassium ions are represented in large black spheres.