Copper Chaperone CupA and Zinc Control CopY Regulation of the Pneumococcal cop Operon

Abstract

As mechanisms of copper toxicity are emerging, bacterial processing of intracellular copper, specifically inside Streptococcus pneumoniae, remains unclear. In this study, we investigated two proteins encoded by the copper export operon: the repressor, CopY, and the copper chaperone, CupA. Zinc suppressed transcription of the copper export operon by increasing the affinity of CopY for DNA. Furthermore, CupA was able to chelate copper from CopY not bound to DNA and reduce it from Cu2+ to Cu1+. This reduced copper state is essential for bacterial copper export via CopA. In view of the fact that innate immune cells use copper to kill pathogenic bacteria, understanding the mechanisms of copper export could expose new small-molecule therapeutic targets that could work synergistically with copper against pathogenic bacteria.

Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY

“…Occurring in species such as Lactococcus lactis and S . pneumoniae (CopR/Y), and in Listeria monocytogenes and Mycobacterium tuberculosis (CsoR), the cop operon repressors remain bound to DNA in environments lacking copper stress to block transcription, and release DNA upon binding copper (16, 24, 27, 32-34).…”

“…These motifs can also bind zinc with a stoichiometry of two CxC motifs needed to bind one zinc (33). Copper binding causes a conformational change in the copper repressor leading to the DNA-binding release of what was thought to be the full cop operon operator, T/GACAnnTGTA (where n represents any nucleotide), while zinc binding leads to tighter cop operon operator binding (32, 33). However, the atomic and protein structural detail of how binding metal directly leads to the conformational changes associated with DNA binding is currently unknown.…”

“…Multiple studies regarding the cop operon have been performed in S . pneumoniae (11, 17, 27, 32, 33, 35, 36). The pneumococcal cop operon contains, copY as the repressor, cupA as a membrane-associated copper chaperone, and copA as the copper-specific exporter (16, 27, 32).…”

“…pneumoniae (11, 17, 27, 32, 33, 35, 36). The pneumococcal cop operon contains, copY as the repressor, cupA as a membrane-associated copper chaperone, and copA as the copper-specific exporter (16, 27, 32). Although the affinities of CopY/R family repressors for copper are generally high, the pneumococcal chaperone CupA is able to chelate copper from CopY, reduce it from Cu 2+ to Cu 1+ , and transport copper to CopA for export (32, 35-37).…”

“…CupA copper chelation allows for the recycling of CopY and its apo- or zinc-bound form to return to the cop operator to repress the operon. Pneumococcal CopY is homologous to several known antibiotic resistance repressors including BlaI, a Staphylococcus aureus MecI homolog that represses the gene for a β - lactamase (32, 33, 38). Like CopY, BlaI and MecI interact with a known operator sequence, TACA/TGTA, form a homodimer, and are mostly helical in secondary structure (32, 38).…”