Mechanisms of zinc binding to the solute-binding protein AztC and transfer from the metallochaperone AztD

Abstract

Bacteria can acquire the essential metal zinc from extremely zinc-limited environments by using ATP-binding cassette (ABC) transporters. These transporters are critical virulence factors, relying on specific and high-affinity binding of zinc by a periplasmic solute-binding protein (SBP). As such, the mechanisms of zinc binding and release among bacterial SBPs are of considerable interest as antibacterial drug targets. Zinc SBPs are characterized by a flexible loop near the high-affinity zinc-binding site. The function of this structure is not always clear, and its flexibility has thus far prevented structural characterization by X-ray crystallography. Here, we present intact structures for the zinc-specific SBP AztC from the bacterium Paracoccus denitrificans in the zinc-bound and apo-states. A comparison of these structures revealed that zinc loss prompts significant structural rearrangements, mediated by the formation of a sodium-binding site in the apo-structure. We further show that the AztC flexible loop has no impact on zinc-binding affinity, stoichiometry, or protein structure, yet is essential for zinc transfer from the metallochaperone AztD. We also found that 3 His residues in the loop appear to temporarily coordinate zinc and then convey it to the high-affinity binding site. Thus, mutation of any of these residues to Ala abrogated zinc transfer from AztD. Our structural and mechanistic findings conclusively identify a role for the AztC flexible loop in zinc acquisition from the metallochaperone AztD, yielding critical insights into metal binding by AztC from both solution and AztD. These proteins are highly conserved in human pathogens, making this work potentially useful for the development of novel antibiotics.

Keywords: ABC transporter; chap; chaperone; crystal structure; metal homeostasis; transport metal; zinc.

Figure 1.

A portion of the complete multiple sequence alignment of cluster A-I SBPs highlighting the flexible loop region. Organism abbreviations are Ecoli, Escherichia coli; Sent, Salmonella enterica; Pden, Paracoccus denitrificans; Syn, Synechocystis 6803; and Spne, Streptococcus pneumoniae. Flexible loop regions as determined by the absence of electron density in crystal structures 2OSV (41) (E. coli ZnuA), 2XY4 (42) (S. enterica ZnuA), 1PQ4 (43) (Synechocystis ZnuA), and 3CX3 (8) (Synechocystis MntC) are shown in red letters. Predicted flexible loop regions are shown in green letters. Residues deleted in the ΔD-loop mutant of P. denitrificans AztC are underlined. Subgroup designations as described in the Introduction and in Table 1 are indicated on the right. The His and Trp residues, indicated by asterisks, are found in the metal-binding site and are absolutely conserved among cluster A-I SBPs.

Figure 2.

Structures of holo- and apoAztC. A and B, crystallographic dimer of holoAztC (A) and apoAztC (B) highlighting interchain contacts mediated by the D-loop (red) and helix 7 (α7). C and D, monomeric structure of the B-chain of holoAztC (C) and the A-chain of apoAztC (D). Backbone structures are shown schematically, with the D-loop colored in red and the Z-loop in magenta. Zinc is shown as a gray sphere and sodium as a purple sphere. The C and N termini are indicated by CT and NT, respectively.

Figure 3.

Structures of the aligned B-chains of holoAztC (green) and apoAztC (blue). The zinc ion in the holo-structure is shown as a gray sphere, and the D-loop of holoAztC has been omitted for clarity.

Figure 4.

The zinc- and sodium-binding sites of holo- and apoAztC. A and B, holoAztC highlighting zinc and its ligands. C and D, apoAztC highlighting former zinc ligands and sodium ligands. E and F, closer view of the sodium-binding site for the B-chain (E) and A-chain (F) of apoAztC. Electron density for a composite omit map at 1.0 σ is shown as blue mesh, and anomalous difference density at 5.0 σ is shown as orange mesh. Hydrogen bond or metal–ligand interactions are shown as dotted black lines. Metal ions are shown as spheres colored according to the element.

Figure 5.

Structural differences between apoAztC (blue) and holoAztC (green). Metal ligand residues are shown as sticks, and metals are shown as spheres colored according to the element. Important secondary structures are also indicated.

Figure 6.

Structure of the D-loop of holoAztC B-chain. A, electron density for a composite omit map at 1.0 σ is shown as blue mesh around residues 116–132. B, interaction between the flexible loop of holoAztC B-chain (green) with surface residues of the A-chain (yellow-green). Specific loop residues and zinc ligands are shown as sticks colored according to element. Zinc is shown as a gray sphere. Hydrogen bond interactions are shown as dotted black lines.

Figure 7.

Structure of ΔD-loop AztC. A, the zinc-binding site and truncated D-loop. Electron density for a composite omit map at 1.0 σ is shown as blue mesh, and anomalous difference density at 5.0 σ is shown as orange mesh. Residues of the truncated loop and zinc ligands are shown as sticks colored according to element. Zinc is shown as a gray sphere. B, overlay of both chains of holo-WT (green and yellow-green) and ΔD-loop (magenta and pink) AztC shown schematically.

Figure 8.

Zinc binding by AztC loop deletion mutants. A, example of a MF2 assay containing 0.5 μm MF2 and 1.0 μm apoAztC. Arrows indicate the direction of fluorescence changes upon titration with increasing zinc. B and C, intensity change at 330 nm with increasing zinc in the absence (filled circles) and presence (empty circles) of ΔD-loop (B) and ΔZ-loop (C) apoAztC. Titrations containing AztC were performed in triplicate, and error bars represent standard error between experiments. Least-squared fits are shown as solid lines.

Figure 9.

Intrinsic fluorescence of zinc binding and transfer from AztD. Fluorescence emission (λ_ex = 278 nm) of 10 μm apoAztC mutants titrated with 2 μm additions of ZnCl₂ (left two columns) or holoAztD (right two columns). Fluorescence intensity increases up to saturation at ∼1 equivalent of added zinc. Titrations with holoAztD (solid lines, increasing in intensity) were followed by the addition of 20 μm ZnCl₂ to determine saturation (dashed line). Each plot is representative of an experiment performed at least twice.

Figure 10.

Proposed mechanism of zinc binding by AztC using apo- (blue) and holo-structures (green). Straight red arrows indicate motions of secondary structures, and curved red arrows indicate motions of side chains labeled in red, going from apo- to zinc-bound. Zinc is shown as a gray sphere and sodium as a purple sphere.