Distinct functions for the flexible loops of zinc specific solute binding proteins from Paracoccus denitrificans

Abstract

In bacteria, ATP binding cassette (ABC) transporters of cluster A‐I exist for the acquisition of the essential trace element zinc from limiting environments. These transporters are crucial for bacterial survival, and the gene knockout strains often demonstrate a pronounced growth defect in minimal media and attenuated virulence in the case of pathogens. Thus such systems are attractive targets for the development of novel antibiotics. Zinc binding with high affinity and specificity is mediated by the solute binding protein (SBP) component of ABC transporters. Zinc‐specific SBPs are characterized by the presence of a flexible loop near zinc binding pocket of the protein. P. denitrificans encodes two zinc ABC transporter operons, znuABC and aztABCD. The SBPs for these systems (AztC and ZnuA) are distinct in the length and sequence of flexible loop regions. The function of the flexible loop in each protein was investigated using fluorescence binding assays and structural methods for WT and loop mutant proteins. The flexible loop of AztC has no impact on zinc binding affinity, stoichiometry or protein structure, yet it is essential for zinc transfer from the metallochaperone AztD. Site specific mutagenesis was employed to evaluate the roles of specific residues in this process and the results showed that three His residues in the loop temporarily coordinate zinc and then convey it to the high‐affinity binding site. Consistent with this, mutation of any of these residues to Ala abrogated zinc transfer from AztD. AztD does not transfer zinc to ZnuA, despite the fact that ZnuA also contains a flexible loop rich in His residues. The ZnuA flexible loop contains 15 His, 11 Glu and 3 Asp residues, providing multiple additional zinc binding sites. In fact, P. denitrificans ZnuA binds up to 6 zinc ions, the most for any zinc SBP studied to date. The results demonstrate distinct roles for zinc SBP flexible loops depending on loop structure and context, potentially allowing for acquisition of zinc from different environments.Support or Funding InformationNational Institute of General Medical Science of the National Institutes of Health under award number 1SC2GM111170‐01.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.