Metal Complexes of Two Specific Regions of ZnuA, a Periplasmic Zinc(II) Transporter from Escherichia coli

Abstract

The crystal structure of ZnZnuA from Escherichia coli reveals two metal binding sites. (i) The primary binding site, His143, is located close the His-rich loop (residues 116-138) and plays a significant role in Zn(II) acquisition. (ii) The secondary binding site involves His224. In this work, we focus on understanding the interactions of two metal ions, Zn(II) and Cu(II), with two regions of ZnuA, which are possible anchoring sites for Zn(II): Ac-₁₁₅MKSIHGDDDDHDHAEKSDEDHHHGDFNMHLW₁₄₅-NH₂ (primary metal binding site) and Ac-₂₂₃GHFTVNPEIQPGAQRLHE₂₄₀-NH₂ (secondary metal binding site). The histidine-rich loop (residues 116-138) has a role in the capture of zinc(II), which is then further delivered into other regions of the protein. For both Zn(II) complexes, histidine residues constitute the main anchoring donors. In the longer, His-rich fragment, a tetrahedral complex with four His residues is formed, while in the second ligand, two imidazole nitrogens are involved in zinc(II) binding. In both cases, so-called loop structures are formed. One consists of a ₁₂₅HxHxExxxExHxH₁₃₇ motif with seven amino acid residues in the loop between the two central histidines, while the other is formed by a ₂₂₄HFTVNPEIQPGAQRLH₂₃₉ motif with 14 amino acid residues in the loop between the two nearest coordinating histidines. The number of available imidazoles also strongly affects the structure of copper(II) complexes; the more histidines in the studied region, the higher the pH in which amide nitrogens will participate in Cu(II) binding.

Figure 1

Representative distribution diagram for formation of the Ac-₂₂₃GHFTVNPEIQPGAQRLHE₂₄₀-NH₂ complex with Cu²⁺ ion at 298 K and I = 0.1 M (NaClO₄). [L] = 0.0005 M; M:L molar ratio of 1:1.

Figure 2

Superimposition of selected regions of one-dimensional ¹H NMR spectra of the Ac-₂₂₃GHFTVNPEIQPGAQRLHE₂₄₀-NH₂ fragment at 1.0 mM in the absence (black) and presence of 0.5 equiv of Zn(II) (blue). The shifts are indicated by arrows.

Figure 3

Zn(II)-induced chemical shift variation of the selected proton of the Ac-₂₂₃GHFTVNPEIQPGAQRLHE₂₄₀-NH₂ fragment at 1.0 mM and pH 7.2 in the presence of 0.5 equiv of Zn(II).

Figure 4

Superimposition of amide regions of (A) one-dimensional ¹H NMR spectra of the Ac-₂₂₃GHFTVNPEIQPGAQRLHE₂₄₀-NH₂ fragment at 1.0 mM in the absence (black) and presence of 0.5 equiv of Zn(II) (blue) at pH 7.2 and (B) ¹H–¹H TOCSY 2D NMR spectra of the ZnuA fragment at 1.0 mM in the absence (black) and pr esence of 0.5 equiv of Zn(II) (magenta) at pH 7.2.

Figure 5

Amide region of ¹H–¹H NOESY 2D NMR spectra of the Ac-₂₂₃GHFTVNPEIQPGAQRLHE₂₄₀-NH₂ fragment at 1.0 mM in the presence of 0.5 equiv Zn(II) at pH 7.2.

Figure 6

Representative distribution diagram for the formation of the complex of Ac-₁₁₅MKSIHGDDDDHDHAEKSDEDHHHGDFNMHLW₁₄₅-NH₂ with a Cu²⁺ ion at 298 K and I = 0.1 M (NaClO₄). [L] = 0.0005 M; M:L molar ratio of 1:1.

Figure 7

Superimposition of selected regions of 2D ¹H–¹H NMR TOCSY spectra of the Ac-₁₁₅MKSIHGDDDDHDHAEKSDEDHHHGDFNMHLW₁₄₅-NH₂ fragment at 1.0 mM in the absence (black) and presence of 0.5 equiv of Zn(II) (magenta) at pH 6.0.

Figure 8

Superimposition of selected amide regions of 2D ¹H–¹H NMR TOCSY spectra of the Ac-₁₁₅MKSIHGDDDDHDHAEKSDEDHHHGDFNMHLW₁₄₅-NH₂ fragment at 1.0 mM in the absence (black) and presence of 0.5 equiv of Zn(II) (magenta) at pH 6.0.

Figure 9

Competition plots for (A) Zn(II)-Ac-MKSIHGDDDDHDHAEKSDEDHHHGDFNMHLW-NH₂ and Zn(II)-Ac-GHFTVNPEIQPGAQRLHE-NH₂ and (B) Cu(II)-Ac-MKSIHGDDDDHDHAEKSDEDHHHGDFNMHLW-NH₂ and Cu(II)-Ac-GHFTVNPEIQPGAQRLHE-NH₂. Previously calculated stability constants are applied to a theoretical situation in which equimolar amounts of Zn(II), Cu(II), and both ligands are present.