A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity

Abstract

Zinc is essential for many cellular processes, including DNA synthesis, transcription, and translation, but excess can be toxic. A zinc-induced gene, smtA, is required for normal zinc-tolerance in the cyanobacterium Synechococcus PCC 7942. Here we report that the protein SmtA contains a cleft lined with Cys-sulfur and His-imidazole ligands that binds four zinc ions in a Zn(4)Cys(9)His(2) cluster. The thiolate sulfurs of five Cys ligands provide bridges between the two ZnCys(4) and two ZnCys(3)His sites, giving two fused six-membered rings with distorted boat conformations. The inorganic core strongly resembles the Zn(4)Cys(11) cluster of mammalian metallothionein, despite different amino acid sequences, a different linear order of the ligands, and presence of histidine ligands. Also, SmtA contains elements of secondary structure not found in metallothioneins. One of the two Cys(4)-coordinated zinc ions in SmtA readily exchanges with exogenous metal ((111)Cd), whereas the other is inert. The thiolate sulfur ligands bound to zinc in this site are buried within the protein. Regions of beta-strand and alpha-helix surround the inert site to form a zinc finger resembling the zinc fingers in GATA and LIM-domain proteins. Eukaryotic zinc fingers interact specifically with other proteins or DNA and an analogous interaction can therefore be anticipated for prokaryotic zinc fingers. SmtA now provides structural proof for the existence of zinc fingers in prokaryotes, and sequences related to the zinc finger motif can be identified in several bacterial genomes.

Figure 1

Sequence and backbone structure of SmtA. (a) Sequence alignment of SmtA homologues from various strains of Synechococcus (S.), Anabaena (A.), Pseudomonas aeruginosa (P.a.), and P. putida (P.p.). Green, conserved residue; yellow, semiconserved residue. The sequences for the Pseudomonads are truncated at the C terminus. (b) “Sausage” representation of a superposition of the backbone traces (N, Cα, C) of the 20 best NMR-derived structures of holo-SmtA without specific incorporation of Zn²⁺. The cylindrical rod represents a trace through the Cα atoms of the protein. Its radius is proportional to the mean global backbone displacement per residue. The rmsd about the mean coordinates for residues 5–56 is 1.36 ± 0.35 Å for a best fit of the backbone atoms.

Figure 2

Proton-decoupled one-dimensional ¹¹¹Cd NMR spectra of SmtA. (a) ¹¹¹Cd₄SmtA prepared by reconstitution of apo-SmtA with ¹¹¹Cd²⁺ (106.04 MHz, pH 7.0, 318 K, 10% D₂O, 50 mM Tris⋅HCl, 50 mM NaCl) showing four peaks with equal integrals. (b) Substitution of Zn²⁺ by Cd²⁺. Spectra recorded after mixing Zn₄SmtA with 1 (bottom), 4 (middle), and 8 (top) mol equivalents of ¹¹¹Cd²⁺ (pH 7.0, 308 K, 10% D₂O, 50 mM Tris⋅HCl, 50 mM NaCl). Comparison with a shows that the first mol equivalent of ¹¹¹Cd²⁺ selectively occupies binding site B, and that binding site A is not occupied at all. The various peaks reflect ¹¹¹Cd²⁺ in mixed Cd, Zn clusters. The occupation of the sites is depicted by squares and circles in the order ABDC, clockwise from top left. Filled square, Cd in the site which gives rise to the respective peak; filled circle, Cd in an adjacent site; open circle, Zn; e.g., the peak at 656 ppm corresponds to ¹¹¹Cd²⁺ in site B (■), with Zn²⁺ in sites A and C (○) and ¹¹¹Cd²⁺ in site D (●). (c) Metal-to-ligand connectivities for SmtA (as determined by 2D heteronuclear NMR experiments).

Figure 3

Features of the NMR solution structure of bacterial Zn₄SmtA and comparison with mammalian MT-2. (a) Energy-minimized average structure of Zn₄SmtA including connectivities for the Zn²⁺ ions. The structure is derived from the 20 best structures. (b) Structure of the [Cd₄] α-domain of rat MT-2 (32) showing the similarity between the inorganic cores of SmtA and the α-domain of mammalian MTs.

Figure 4

(a) Weak interactions around binding site A: (i) CH/π interaction between the β-sheet and the α-helix. The distance between H(α) of Ala-37 and the aromatic ring of Tyr-31 is 2.47 Å. (ii) Hydrogen bond between NH of Cys-32 and the sulfur of Cys-9 (2.61 Å, 153°). (b) Overlay of site A in SmtA with the zinc site in a DNA-binding domain of the eukaryotic transcription factor GATA-1 (41). Overlay of all side chain atoms of the Cys residues results in a rmsd of 1.40 Å. (c) Sequence alignment of the molecules shown in b. Arrows and curls indicate elements of secondary structure. The only conserved residues are the binding site Cys, the Gly in the loop between the β-strands, and an Ala residue adjacent to Cys-36 in SmtA.