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

Abstract

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. 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 b 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.

AztD, a Periplasmic Zinc Metallochaperone to an ATP-binding Cassette (ABC) Transporter System in Paracoccus denitrificans

“…The periplasmic protein ZinT has also been implicated in maintaining zinc homeostasis in the periplasm, although its precise role is still unclear. The most recent results suggest that ZinT functions in conjunction with ZnuABC to import zinc, perhaps by maximizing efficient transfer of zinc to ZnuA (17)(18)(19)(20). In support of this hypothesis, ZinT forms a complex with ZnuA in vitro but only in the metallated form (19,20).…”

“…8). A detailed biophysical investigation of the ZnuA-ZinT complex combining x-ray crystallography, analytical ultracentrifugation, and x-ray scattering spectroscopy suggested that the His-rich loop of ZnuA is required for complex formation and that it fits into a hydrophobic cleft leading to the zinc site of ZinT (20). These data suggested a model of metal transfer from ZinT to ZnuA requiring the participation of metal ligands on the His-rich loop.…”

“…However, there are several key differences between the two systems. ZinT and AztD are completely unrelated by primary sequence and must adopt completely different folds as ZinT has significant ␣-helical content (20) although AztD has virtually none on the basis of CD spectroscopy (Fig. 4).…”

The First Histidine Triad Motif of PhtD Is Critical for Zinc Homeostasis in Streptococcus pneumoniae

“…PhtD, the most highly conserved Pht protein across pneumococcal strains, contains five His triad (HT) motifs, and each motif is thought to bind one Zn 2ϩ ion (8). Loisel et al produced a truncated derivative of PhtD containing only one HT motif and determined the affinity of the fragment for Zn 2ϩ to be 131 Ϯ 10 nM, which is weaker than that of AdcA (2.4 Ϯ 0.1 nM) and other Zn 2ϩ -binding cluster A-I SBPs (4,8,16). This is consistent with the model for Zn 2ϩ transfer to the higher-affinity site of a Zn 2ϩ -binding SBP for transport into the bacterial cell.…”

Conservative interpretation of small-angle X-ray scattering data from biological macromolecules

“…This suggests that the inter-domain contacts observed in the crystal are not present in solution, and that the two domains move independent of one another, attached by a flexible linker. This differs from previous solution studies on S. enterica AdcA, which found that the separate ZinT and ZnuA domains formed a stable interaction (55). Our data suggests that in S. pneumoniae AdcA, any such interaction is transient, but does not preclude the possibility that they interact in specific circumstances such as Zinc shortage.…”