title: 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: 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 KD value of 22±2nM for ZinT, while those with ZnuA point to one high affinity (KD<20nM) 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.
  
  
  CONCLUSIONS
  Knowledge of the ZinT-ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.
authors.affiliation: CNR Institute of Molecular Biology and Pathology and Department of Biochemical Sciences, "Sapienza" University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Ilari
authors.forename: Flaminia
authors.initials: F
authors.lastname: Alaleona
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Tria
authors.forename: Patrizia
authors.initials: P
authors.lastname: Petrarca
authors.forename: Andrea
authors.initials: A
authors.lastname: Battistoni
authors.forename: Carlotta
authors.initials: C
authors.lastname: Zamparelli
authors.forename: Daniela
authors.initials: D
authors.lastname: Verzili
authors.forename: Mattia
authors.initials: M
authors.lastname: Falconi
authors.forename: Emilia
authors.initials: E
authors.lastname: Chiancone
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019897:Periplasm / Q000378:metabolism*; D011487:Protein Conformation; D019779:Salmonella enterica / Q000378:metabolism*; D053838:Scattering, Small Angle; D017386:Sequence Homology, Amino Acid; D014461:Ultracentrifugation; D014961:X-Ray Diffraction; D015032:Zinc / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D015032:Zinc
keywords: Analytical ultracentrifugation; Protein-protein interaction; SAXS; Salmonella enterica; Zinc affinity; Zinc transport