The Salmonella enterica ZinT structure, zinc affinity and interaction with the high-affinity Uptake protein ZnuA provide insight into the management of periplasmic zinc

Background: In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT-ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim. Methods: ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy). Results: The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a K-D value of 22 2 nM for ZinT, while those with ZnuA point to one high affinity (K-D <20 nM) and one low affinity Zn(II) binding site (KD in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT-ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other. Conclusions: ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA. General significance: Knowledge of the ZinT-ZnuA relationship is crucial for understanding bacterial Zn(II) uptake. (c) 2013 Elsevier B.V. All rights reserved.