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Metal binding specificity in a putative nickel solute binding protein.
Author(s) -
Amato Matthew,
Yukl Erik
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.652.42
Subject(s) - operon , atp binding cassette transporter , periplasmic space , biochemistry , transporter , binding site , cloning (programming) , chemistry , zinc , biology , gene , escherichia coli , organic chemistry , computer science , programming language
Researchers are pressured to identify novel antibiotic targets in response to the epidemic of bacterial antibiotic resistance. Transition metal homeostasis is related to pathogenicity in gram‐negative bacteria. In particular, metal transporters of the ATP binding cassette (ABC) transporter family are essential for acquisition of specific metals in highly limited environments such as the human host. These systems depend upon a periplasmic solute binding protein (SBP) to bind the appropriate metal with high affinity and specificity. Transcriptional analysis in our model gram‐negative organism Paracoccus denitrificans showed three operons of note encoding ABC transporters to be upregulated under zinc deprivation. Two of these, aztABCD and znuABC, belong to the cluster 1‐A family, which is predicted to transport zinc. Further, the solute binding proteins (AztC and ZnuA) have been shown to bind extracellular zinc with high affinity and specificity in vitro . The third operon pden1341‐1345 encodes a cluster 5 ABC transporter system predicted to be a Nickel/di‐peptide transporter. Thus, it is perplexing that this operon should be significantly upregulated under conditions of zinc deprivation. To determine the possible metal substrate for the Pden1341‐1345 operon, the SBP Pden1341 was cloned, expressed, purified and binding to various metals was determined. Traditional cloning techniques failed by what is thought to be a complication of its large gene size. Gibson cloning proved an effective alternative to cloning full‐length pden1341 . Anion exchange and size exclusion chromatography were used to purify Pden1341, and ICP‐OES analysis suggested Pden1341 purifies without bound metal. Binding to various metals was determined by a competitive fluorescence assay as well as isothermal titration calorimetry. The results of these experiments suggest alternate roles for the cluster 5 ABC transports beside the transport of nickel or dipeptides. Thus, cluster 5 ABC transporters may play unexpected functions in metal homeostasis among gram‐negative organisms that may be relevant to the development of metal transport inhibitors as novel antibiotics. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .