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 (K_D 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.