The Role of a Flexible loop in Metal transfer between periplasmic zinc proteins

Abstract

Zinc is an essential nutrient, and proteins mediating zinc acquisition 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. Using a simple fluorescence assay, we have found that the zinc SBP AztC can acquire zinc from solution or from a metallochaperone, AztD. The crystal structure of AztC shows that, like all known zinc‐specific SBPs, this protein has a flexible loop near the zinc binding site. We hypothesized that this flexible loop is required for the recognition and subsequent transfer of zinc from its chaperone AztD. To test this, we generated a deletion mutant lacking the flexible loop (Δloop AztC) and tested zinc acquisition from solution and from AztD. Consistent with our hypothesis, the affinity of the mutant protein for zinc in solution is unaffected, yet it can no longer acquire zinc from AztD. In order to refine our understanding of those residues required for AztD recognition and zinc transfer, several site directed mutants of conserved loop residues were generated and evaluated for their ability to acquire zinc from AztD. Three His residues appeared to be absolutely required for zinc transfer, while mutation of a conserved Asp had no effect. Tyr121 appeared to be dispensable for zinc transfer, although the process was significantly slower for the Y121A mutant relative to WT. A detailed understanding of the mechanisms governing extracellular zinc management in bacteria may eventually lead to the development of novel antibiotics targeting this important process.Support or Funding InformationNIH (National Institutes of Health) grant number: 1sc2gm111170‐01