Insight into the interaction of metal ions with TroA from Streptococcus suis

Abstract

Background: The scavenging ability of sufficient divalent metal ions is pivotal for pathogenic bacteria to survive in the host. ATP-binding cassette (ABC)-type metal transporters provide a considerable amount of different transition metals for bacterial growth. TroA is a substrate binding protein for uptake of multiple metal ions. However, the function and structure of the TroA homologue from the epidemic Streptococcus suis isolates (SsTroA) have not been characterized.

Methodology/principal findings: Here we determined the crystal structure of SsTroA from a highly pathogenic streptococcal toxic shock syndrome (STSS)-causing Streptococcus suis in complex with zinc. Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed that apo-SsTroA binds Zn(2+) and Mn(2+). Both metals bind to SsTroA with nanomolar affinity and stabilize the protein against thermal unfolding. Zn(2+) and Mn(2+) induce distinct conformational changes in SsTroA compared with the apo form as confirmed by both circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra. NMR data also revealed that Zn(2+)/Mn(2+) bind to SsTroA in either the same site or an adjacent region. Finally, we found that the folding of the metal-bound protein is more compact than the corresponding apoprotein.

Conclusions/significance: Our findings reveal a mechanism for uptake of metal ions in S. suis and this mechanism provides a reasonable explanation as to how SsTroA operates in metal transport.

Figure 1. Genetic organization of the S. suis Tro operon and alignment of SsTroA homologues.

(A) Gene descriptions of the S. suis Tro operon. (B) Structure-based multiple sequence alignment of SsTroA homologues. The most-conserved residues in all homologues are shaded blue. The four metal-coordinating residues are highlighted in green. The LXXC lipoprotein motif (type II signal peptides for Sec-dependent transport) is highlighted in red. The two conserved residues predicted to form the salt bridge are marked in yellow. Secondary structure elements for SsTroA are shown above the sequence. TpTroA, TroA from Treponema pallidum; SpPsaA, PsaA from Streptococcus pneumonia; SyMntC, MntC from Synechocytis 6803.

Figure 2. ITC analysis of the SsTroA interaction with Zn and Mn.

Left: apo-SsTroA (90 µM) with addition of Zn²⁺ (500 µM); Right: apo-SsTroA (30 µM) with Mn²⁺ (200 µM). In each case, the upper panel shows raw energy changes during the titration, while the lower panel presents the integrated peak areas. The fitting of the data yielded the thermodynamic parameters listed in Table S2.

Figure 3. X-band EPR spectra and ANS fluorescence analysis of SsTroA.

(A) EPR spectra of Mn²⁺ (100 µM) in the absence (red line) and presence (purple line) of SsTroA (100 µM) at room temperature in solution. The total Mn²⁺ concentration was the same in both samples. The signal intensity decrease corresponds to the zero-field splitting arising from the disturbances in the octahedral ligand field of the bound metal. The instrument conditions are as follows: microwave power, 20 mW; microwave frequency, 9.53 GHz; modulation frequency, 100 KHz; modulation amplitude, 1 G; and modulation amplitude, 1.0 mT. (B) Fluorescence changes in the intensity emission for SsTroA indicate a metal dependent decrease in hydrophobic residue exposure. Emission spectra of ANS in the presence of different SsTroA states: apo-SsTroA (purple), Zn²⁺-SsTroA (green) and Mn²⁺-SsTroA (red). A significant enhancement in fluorescence is probed on binding of ANS to apo-SsTroA. This fluorescence is slightly diminished in the presence of metal ions, which indicates the ordering of the metal binding domain of the SsTroA structure when metal is bound.

Figure 4. CD spectra of SsTroA.

(A) Metal ions induced conformational changes monitored by far-UV CD spectra. Far-UV CD spectra were acquired for apo-SsTroA (purple) and SsTroA in the presence of 100 µM Zn²⁺ (green), or 100 µM Mn²⁺ (red). (B) Thermal unfolding followed by far-UV CD spectra at 223 nm. Data for apo-SsTroA (purple) and SsTroA in the presence of 100 µM Zn²⁺ (green) or 100 µM Mn²⁺ (red).

Figure 5. Crystallographic structure of SsTroA and structural comparisons of SsTroA with structurally known Mn-specific SBPs.

(A) Cartoon diagram of the SsTroA, the N-terminal domain is shown as red, the C-terminal domain is green, and the linking helix is colored purple. The residues forming the metal binding site are presented in sticks-balls format, two conserved Glu residues predicted to form the salt bridge are displayed in cyan, and the zinc ion is displayed as a lightpink sphere. The α-helices are designated α1–α9, and the β strands are β1–β8. (B) The coordination bonds formed between the Zn²⁺ and His 76, His 139, His 205 and Asp 289. The distances between Zn²⁺ and these residues are calculated. (C) Bottom view of the structural comparison. (D) Side view of the comparison. Comparisons shown here are SsTroA (purple), TpTroA (marine), and S. pneumoniae PsaA (limegreen). The unique loops are designated as Loop 1 and Loop 2. Structures are displayed in ribbon representation.

Figure 6. NMR spectra of SsTroA complexed with Zn²⁺ or Mn²⁺.

(A) Superimposition of two-dimensional ¹H¹⁵N-HSQC spectra comparing 0.5 mM apo-SsTroA (purple) with 0.5 mM Zn²⁺-SsTroA (green). (B) Overlay of 2D HSQC spectra comparing 0.5 mM apo-SsTroA (purple) with 0.5 mM Mn²⁺-SsTroA (red). (C) Comparison of 0.5 mM Zn²⁺-SsTroA (green) with 0.5 mM Mn²⁺-SsTroA (red). All spectra were acquired at 25°C in 20 mM sodium acetate (pH 6.5), and spectra were recorded at a 600-MHz ¹H frequency. A number of the cross-peaks exhibiting significant shifts are highlighted in circles, indicating local conformational changes in the metal binding pocket.

Figure 7. Hypothetical model of SsTroA participation in the metal ion homeostasis of S. suis.

SsTroA is a substrate binding protein, which is anchored into the membrane via a lipid-anchor. It feeds the ligand into the translocation pathway formed by the SsTroC and SsTroD. The nucleotide binding domains (SsTroB) hydrolyze ATP to drive the transport of the ligand through the membrane.