Crystal Structures of the Periplasmic Metallochaperone AztD

Abstract

High efficiency zinc import through ATP binding cassette (ABC) transporters is critical for the survival of many bacteria in zinc limited conditions. These systems are especially important for human pathogens due to severe zinc depletion at the site of infection as a result of “nutritional immunity”. Thus, bacterial ABC transporters for zinc are promising targets for the development of novel antibiotics. These systems are composed of a transmembrane permease, cytoplasmic ATPase and periplasmic or lipoprotein solute binding protein (SBP). The latter is required to bind zinc with high affinity and specificity and deliver it to the membrane permease for import into the cytoplasm. Recent data also indicates the participation of other periplasmic zinc-binding proteins in metal import through ABC transporters in some species. We have recently identified a zinc ABC transporter operon (aztABCD) in Paracoccus denitrificans that encodes a periplasmic metallochaperone (AztD) capable of transferring zinc to the associated SBP (AztC) through a specific, associative mechanism. The process can be followed in vitro using the significant increase of tryptophan fluorescence in AztC upon zinc binding. The aztABCD operon is highly conserved in several human pathogens, including the carbapenem-resistant Enterobacteriaceae (CRE) species Citrobacter koseri. We have solved the crystal structures of P. denitrificans and C. koseri AztD using single wavelength anomalous diffraction (SAD). These proteins adopt a beta-propeller fold that has never before been associated with metal transfer functions. Two highly conserved zinc binding sites were identified. Fluorescence zinc transfer assays with mutant AztD constructs indicate that only one of these sites transfers zinc to AztC. Protein-protein docking of AztC and AztD structures identify interaction interfaces and residue contacts essential for metal transfer that were again confirmed by mutagenesis. These data provide structural data on a completely new family of periplasmic metallochaperones as well as detailed mechanistic information on the process of metal transfer from AztD to AztC. As such, this work may be valuable in the rational design of small molecule inhibitors of metal transfer as antibiotics in multiple drug resistant bacteria such as the CRE. Support or Funding Information Funded by the National Institute of General Medical Science of the National Institutes of Health under award number R01GM122819. Docking of AztC (magenta) and AztD (green) showing the transient zinc transfer intermediate. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.