Physical basis of metal-binding specificity in Escherichia coli NikR

Abstract

In Escherichia coli and other bacteria, nickel uptake is regulated by the transcription factor NikR. Nickel binding at high-affinity sites in E. coli NikR (EcNikR) facilitates EcNikR binding to the nik operon, where it then suppresses transcription of genes encoding the nickel uptake transporter, NikABCDE. A structure of the EcNikR-DNA complex suggests that a second metal-binding site is also present when NikR binds to the nik operon. Moreover, this co-crystal structure raises the question of what metal occupies the second site under physiological conditions: K(+), which is present in the crystal structure, or Ni(2+), which has been proposed to bind to low- as well as high-affinity sites on EcNikR. To determine which ion is preferred at the second metal-binding site and the physical basis for any preference of one ion over another in both the second metal-binding site and the high-affinity sites, we conducted a series of detailed molecular simulations on the EcNikR structure. Simulations that place Ni(2+) at high-affinity sites lead to stable trajectories with realistic ion-ligand distances and geometries, while simulations that place K(+) at these sites lead to conformational changes in the protein that are likely unfavorable for ion binding. By contrast, simulations on the second metal site in the EcNikR-DNA complex lead to stable trajectories with realistic geometries regardless of whether K(+) or Ni(2+) occupies this site. Electrostatic binding free energy calculations, however, suggest that EcNikR binding to DNA is more favorable when the second metal-binding site contains K(+). An analysis of the energetic contributions to the electrostatic binding free energy suggests that, while the interaction between EcNikR and DNA is more favorable when the second site contains Ni(2+), the large desolvation penalty associated with moving Ni(2+) from solution to the relatively buried second site offsets this favorable interaction term. Additional free energy simulations that account for both electrostatic and non-electrostatic effects argue that EcNikR binding to DNA is most favorable when the second site contains a monovalent ion the size of K(+). Taken together, these data suggest that the EcNikR structure is most stable when Ni(2+) occupies high-affinity sites and that EcNikR binding to DNA is more favorable when the second site contains K(+).

Figure 1

Crystal structures of EcNikR and the metal-binding sites. Each of the four monomers is colored individually, and nickel ions are represented with green spheres and potassium ions with purple spheres. (a) Ni²⁺-bound EcNikR (PDB 2HZA) and the square-planar high-affinity nickel-binding site. (b) EcNikR-DNA complex (PDB 2HZV) and the second metal-binding site with potassium bound in an octahedral geometry.

Figure 2

Representative structures of the high-affinity metal-binding sites after energy minimization and MD simulation studies. High-affinity site with either (a) Ni²⁺ or (b) K⁺ after energy minimization. Metal–ligand distances are shown in Å. (c) Average structure from MD simulation of high-affinity site containing K⁺. Coloring is the same as in Figure 1. Water molecules are represented as red spheres.

Figure 3

Representative structures of the second metal-binding site from minimization studies of the EcNikR-DNA complex with either (a) K⁺ or (b) Ni²⁺ bound in the second metal-binding site. Distances are in Å, and coloring is the same as in Figure 1.

Figure 4

Thermodynamic path comparing EcNikR-DNA binding with either K⁺ (ΔG_K) or Ni2+ (ΔG_Ni) at the second metal-binding site. Red rectangles represent the MBD, black triangles the RHH domains, and parallel green lines the DNA, and ions are explicitly shown in blue or orange. The ions not bound to the structure are infinitely distant from the protein.

Figure 5

Thermodynamic cycle for calculating the electrostatic free energy difference associated with replacing potassium with nickel in the second metal-binding site. Numbers in circles delineate each structure in the pathway. Red rectangles represent the MBD, black triangles the RHH domains, and parallel green lines the DNA, and K⁺ ions are explicitly shown in purple. The Ni²⁺ ions are shown in either blue or orange, depending on the geometry of the groups that surround the ion. In structure 3, Ni²⁺ is placed in the secondary site; however, the coordinating species in the site are arranged in a manner that is consistent with a bound K⁺ ion (Ni²⁺ colored blue). In structure 4, the coordinating residues have been energy minimized, yielding a rearranged site that has Ni²⁺-preferred geometry and bond distances (Ni²⁺ colored orange).

Figure 6

EcNikR-DNA structure, with residues with the most extreme contributions to ΔΔG colored in red and blue. Red residues contribute ≤−10 kcal/mol to ΔΔG and prefer Ni²⁺ ions in the second metal-binding sites (black spheres). Residues in blue contribute ≥10 kcal/mol to ΔΔG and prefer K⁺ ions in the second metal-binding sites. All backbone atoms are shown in ribbon form and are colored gray. Residues that contribute between −10 and 10 kcal/mol to the ΔΔG are not shown in stick form.

Figure 7

Thermodynamic cycle for calculating the free energy difference associated with replacing a monovalent cation with the radius of potassium with a cation of larger or smaller radius. NikR representations are the same as in Figure 4, and “X⁺” represents an ion having different Lennard-Jones parameters than K⁺.