Transcriptional Regulation, Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans

Abstract

ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn(2+) and Mn(2+) from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn(2+) or Mn(2+) specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties, and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn(2+) over Mn(2+) specificity, the Zn(2+) ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn(2+)-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn(2+)-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn(2+)-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn(2+) specificity.

Keywords: ABC Transporter; Manganese; Metal Homeostasis; Metalloprotein; Transcription Regulation; Zinc.

FIGURE 1.

Multiple sequence alignments of Pden1597 with putative Mn²⁺ (Pden1259) and Zn²⁺ (Pden4140) SBPs from P. denitrificans, the Mn²⁺ SBP PsaA from Streptococcus pneumoniae (Sp_PsaA) and the Zn²⁺ SBP ZnuA from Salmonella typhimurium (St_ZnuA). Black and gray shading indicate regions of sequence identity and similarity, respectively. Asterisks above the sequences indicate possible positions of metal ligands, whereas residues highlighted in yellow are metal ligands observed in crystal structures. The red box highlights the His-rich loop observed in Zn-specific SBPs.

SCHEME 1

FIGURE 2.

A, growth curves for P. denitrificans in metal-replete (open circles), Zn²⁺-depleted (open squares), Mn²⁺-depleted (crosses), and Zn²⁺-chelated (filled diamonds) media. Data are the average of three independent biological replicates with error bars corresponding to the S.D. between replicates. B, relative expression levels of pden1597 and pden4140 under growth conditions containing 50 μm Zn²⁺ (black bars), 0 μm Zn²⁺ (gray bars), or 0 μm Zn²⁺ in the presence of 50 μm TPEN (white bars). Error bars represent the mean ± S.E. (n = 3). C, relative expression levels of pden1597, pden4140, and pden1259 under growth conditions containing 50 μm Mn²⁺ (black bars) and 0 μm Mn²⁺ (white bars).

FIGURE 3.

A, SDS-PAGE gel showing the purification of Pden1597. Lane 1, M_r ladder; lane 2, total cellular protein before isopropyl 1-thio-β-d-galactopyranoside induction; lane 3, total cellular protein after isopropyl 1-thio-β-d-galactopyranoside induction; lane 4, periplasmic fraction; lane 5, combined fractions containing Pden1597 after anion exchange chromatography; lane 6, combined fractions containing Pden1597 after size exclusion chromatography. B, size exclusion chromatogram showing purification of Pden1597 and the molecular weights calculated from the elution time (calc. MW) and the primary sequence (pred. MW). C, MS/MS spectrum of the 2+ charge state of peptide Glu-25 to Lys-42. The monoisotopic [M + 2H]²⁺ observed precursor m/z value was 947.01. The y- and b-ions identified are mapped onto the primary sequence.

FIGURE 4.

Representative fluorescence excitation spectra of a competition experiments between apo-Pden1597 and MF-2 titrated with Zn²⁺ (A) or Mn²⁺ (C and E). The initial and final spectra are shown as heavy lines and the direction of intensity changes with increasing metal are indicated by arrows. Changes in fluorescence intensity for MF-2 alone (open circles) or in the presence of apo-Pden1597 (filled circles) or Pden1597 as isolated (crosses) were plotted versus [Zn] at 330 nm (B) or [Mn] at 360 nm (D and F) and the competition data were fitted (solid line). The concentration of Pden1597 was 1.0 (A–D) or 15.0 μm (E and F). MF-2 was present at 0.4 (A, B, E, and F) or 0.8 μm (C and D).

FIGURE 5.

A, CD spectra of 15 μm apo-Pden1597 alone (solid black line) and in the presence of 40 μm Mn²⁺ (broken black line) and 40 μm Zn²⁺ (solid gray line). B, melt curve data measured at 223 nm for the samples in panel A, apo-Pden1597 alone (filled black diamonds), and in the presence of 40 μm Mn²⁺ (open black squares) and 40 μm Zn²⁺ (filled gray triangles).

FIGURE 6.

A, Overall structure of Pden1597 showing the N-terminal domain (blue), C-terminal domain (red), and connecting helix (gray) in schematic form. Metal ligands and Cys residues are shown in stick form colored according to atom type and the Zn²⁺ atom is shown as a gray sphere. B and C, the metal binding site of Pden1597. Blue electron density is for the 2F_o − F_c map of the refined structure and is contoured at 1.5 σ. Green density is for the F_o − F_c map of the structure omitting Zn²⁺ and is contoured at 5.0 σ.

FIGURE 7.

Overlay of the structures of Pden1597 (light blue) with that of Zn²⁺-bound TroA (magenta, PDB accession 3MFQ) in schematic form with Zn²⁺-binding residues shown in stick form colored according to atom type. Asterisks indicate the positions of residues beyond which the Pden1597 loop could not be modeled.