Taken together, the structural and functional data we have presented here clearly demonstrate that SsTroA binds both Zn 2+ and Mn 2+ with high affinity. In addition, the binding of metal ions increased the protein stability and induced conformational changes. Specifically, further analysis revealed that Zn 2+ and Mn 2+ bind to the SsTroA in either the same site or very nearby. The result of this work provides novel insight into the S. suis 2 divalent metal uptake process and establishes a framework for understanding the divalent metal homeostasis in S. suis. Additionally, we propose a functional model for the transport of metal ions Figure 6 . NMR spectra of SsTroA complexed with Zn 2+ or Mn 2+ . (A) Superimposition of two-dimensional 1 H 15 N-HSQC spectra comparing 0.5 mM apo-SsTroA (purple) with 0.5 mM Zn 2+ -SsTroA (green). (B) Overlay of 2D HSQC spectra comparing 0.5 mM apo-SsTroA (purple) with 0.5 mM Mn 2+ -SsTroA (red). (C) Comparison of 0.5 mM Zn 2+ -SsTroA (green) with 0.5 mM Mn 2+ -SsTroA (red). All spectra were acquired at 25uC in 20 mM sodium acetate (pH 6.5), and spectra were recorded at a 600-MHz 1 H frequency. A number of the cross-peaks exhibiting significant shifts are highlighted in circles, indicating local conformational changes in the metal binding pocket. doi:10.1371/journal.pone.0019510.g006 by SsTroBCD based upon similar substrate transporters. However, further structural and functional experiments are necessary to provide evidence to support our proposed model. Studies to address these issues are underway, and will lay a solid foundation for future studies of the metal ion transport mechanism.