AztD, a Periplasmic Zinc Metallochaperone to an ATP-binding Cassette (ABC) Transporter System in Paracoccus denitrificans

Abstract

Bacterial ATP-binding cassette (ABC) transporters of transition metals are essential for acquisition of necessary elements from the environment. A large number of Gram-negative bacteria, including human pathogens, have a fourth conserved gene of unknown function adjacent to the canonical permease, ATPase, and solute-binding protein (SBP) genes of the AztABC zinc transporter system. To assess the function of this putative accessory factor (AztD) from Paracoccus denitrificans, we have analyzed its transcriptional regulation, metal binding properties, and interaction with the SBP (AztC). Transcription of the aztD gene is significantly up-regulated under conditions of zinc starvation. Recombinantly expressed AztD purifies with slightly substoichiometric zinc from the periplasm of Escherichia coli and is capable of binding up to three zinc ions with high affinity. Size exclusion chromatography and a simple intrinsic fluorescence assay were used to determine that AztD as isolated is able to transfer bound zinc nearly quantitatively to apo-AztC. Transfer occurs through a direct, associative mechanism that prevents loss of metal to the solvent. These results indicate that AztD is a zinc chaperone to AztC and likely functions to maintain zinc homeostasis through interaction with the AztABC system. This work extends our understanding of periplasmic zinc trafficking and the function of chaperones in this process.

Keywords: ABC transporter; chaperone; metal homeostasis; metalloprotein; zinc.

FIGURE 1.

Portion of the output acquired from the STRING database (44) using AztD as the search query. Homologues to AztD (red), the SBP AztC (purple), the membrane permease AztB (green), and the ATPase AztA (light blue) are shown in their genomic contexts across various bacterial groups.

FIGURE 2.

A, relative expression levels of aztC and aztD under growth conditions containing 50 μm zinc (black bars), 0 μm zinc (gray bars), or 0 μm zinc in the presence of 50 μm TPEN (white bars). Error bars represent the standard error of the mean (n = 3). B, 1% agarose gel of PCR products amplifying across the aztC-aztD gene boundary using a gDNA template (lane 2) or cDNA from cells grown in 50 μm zinc or 0 μm zinc + 50 μm TPEN (lanes 3 and 4, respectively). The expected size of the PCR product is 1157 bp.

FIGURE 3.

A, SDS-polyacrylamide gel. Lanes 1 and 7, molecular weight ladder; lane 2, total cellular protein before IPTG induction; lane 3, total cellular protein after IPTG induction; lane 4, periplasmic fraction; lane 5, combined fractions containing AztD after anion exchange chromatography; lane 6, combined fraction containing AztD after size exclusion chromatography. B, zinc content of AztD after addition of up to 4 molar eq of ZnSO₄. Samples were desalted to remove adventitious metal prior to ICP-OES.

FIGURE 4.

A, CD spectrum of AztD at 25 °C fitted with secondary structure composed of 52% β-sheet, 18% turn, 22% unordered, and 5% α-helix as described under “Experimental Procedures.” B, ellipticity at 215 nm monitored as a function of temperature.

FIGURE 5.

Representative fluorescence excitation spectra of a competition experiment between 15 μm apo-AztD and 0.5 μm MF-2 titrated with zinc (A and B) or manganese (C and D). Changes in fluorescence intensity for MF-2 alone (open circles) or in the presence of apo-AztD (filled circles) were plotted versus [zinc] at 330 nm (B) or [manganese] at 360 nm (D). Zinc competition data were fitted with a three-binding site model as indicated by the solid line in B.

FIGURE 6.

Fluorescence emission spectra using λ_exc = 278 nm for 10 μm apo-AztC (A) and apo-AztD (B) titrated with ZnSO₄.

FIGURE 7.

Fluorescence emission spectra using λ_exc = 278 nm for 10 μm apo-AztC titrated with 2 μm additions of holo-AztD (A), apo-AztD (B), ZnSO₄ in the presence of 1 mm EDTA (E), and holo-AztD in the presence of 1 mm EDTA (F) are shown. Spectra for 10 μm holo-AztC titrated with 2 μm additions of apo-AztD are shown in C with fluorescence change versus [apo-AztD] shown in D. The broken line spectra in A–C are for addition of 20 μm ZnSO₄ following titration to 12 μm holo- or apo-AztD. Arrows indicate the direction of overall fluorescence change during the titration.

FIGURE 8.

Size exclusion chromatograms of holo-AztD (A), apo-AztC (B), and holo-AztD incubated with apo-AztC (C and D) are shown. C is fit with two Gaussian curves representing AztD and AztC individually, allowing quantitation of each protein compared with [zinc] in D. SDS-PAGE and zinc quantitation of individual 1-ml fractions are shown in E.