Resolving the structure of metallochaperone AztD

Abstract

Zinc is an essential nutrient, and proteins mediating zinc homeostasis are important virulence factors among pathogenic bacteria, making them attractive targets for the development of novel antibiotics. Of particular interest are the ATP binding cassette (ABC) transporters, which utilize extracellular solute binding proteins (SBPs) to bind zinc with high affinity and specificity for delivery into the cell via integral membrane permeases. We are studying the zinc import machinery of Paracoccus denitrificans as a model for highly homologous systems in pathogenic bacteria including the carbapenem resistant enterobacteraceae (CRE) species Klebsiella pneumoniae and Citrobacter koseri. Specifically, these species encode an unusual zinc ABC transporter operon aztABCD that encodes a periplasmic metallochaperone (AztD) that transfers zinc specifically to the SBP (AztC). The structure for PdAztC has been solved and features a long, protruding loop containing three His residues. Deletion of the loop or mutation of any of the His residues to Ala completely abrogated zinc transfer from AztD but had no impact on metal binding from solution. From this data, we hypothesized that the loop must be required to access the zinc binding site in AztD, chelate the metal and return it to the AztC binding site. However, without structural information on the AztD chaperone the details of this mechanism remained unclear. We have recently solved high‐resolution crystal structures of AztD from both P. denitrificans and C. koseri using single wavelength anomalous dispersion (SAD) on a lead derivative crystal. This was necessary as there were no homologous of AztD in the protein databank. The protein is a 7‐bladed beta propeller, with zinc bound to three conserved His residues in the central pore. A second zinc binding site was also identified, consistent with solution data indicating that AztD can bind up to three zinc ions. A detailed mechanism for zinc transfer from the primary AztD binding site to AztC is proposed based on the existing solution data, new structures, accessibility of the AztD zinc binding site and electrostatic complementarity between AztD and AztC surfaces. Given the importance of dynamic zinc transfer for bacterial zinc homeostasis and the role of this process in pathogenesis, high‐resolution structural data on zinc binding and transport proteins may be very useful in the development of novel antibiotics for resistant bacteria such as C. koseri.Support or Funding InformationNational Institutes of Health under award number 1SC2GM111170‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.