Thesis (Ph.D.) - Indiana University, Biochemistry, 2015Pneumococcal related disease is an infection caused by Streptococcus pneumoniae (pneumococcus), including but not limited to otitis media, pneumoniae and meningitis. About 40 % of the infections are caused by drug resistant Streptococcus pneumoniae (DRSP). The prevalence of DRSP requires identifying novel targets for fighting against pneumococcal related diseases. The bacterial copper homeostasis machinery emerges as one important determinant for survival and virulence for bacteria in human host and serves as one potential drug development target. One cop operon (copY-cupA-copA) has been demonstrated to be essential for copper resistance in S .pneumoniae cells. The transcription of the cop operon is induced in S. pneumoniae isolated from the lungs and nasopharynx of intranasally infected mice and is required for bacterial growth in nasopharynx. The cop operon encodes three proteins, the copper-specific transcriptional regulator CopY, a protein of unknown function prior to this thesis work CupA and a copper-exporting P1B type ATPase CopA. In my thesis work, CupA has been shown to represent a novel class of copper chaperone widely distributed among lactobacillus and streptococcus, which lack CopZ-like Cu(I) chaperones identified in Enterococcus hirae and Bacillus subtilis. Subcellular cell fractionation experiment confirms that CupA harbors a single N-terminal membrane-spanning helix, thus is the first identified cell membrane-anchored copper chaperone. The membrane association may facilitate copper loading onto CupA as copper gets into the bacterial cell. Cell growth experiments reveal that a Cu(I)-binding competent, membrane-localized CupA is obligatory for bacterial copper resistance. The crystal structures of the soluble domain of CupA (sCupA) and N-terminal MBD of CopA (CopAMBD) in the Cu(I) bound state reveal isostructural cupredoxin-like folds each harboring a binuclear Cu(I) cluster unprecedented in bacterial copper trafficking. NMR studies reveal unidirectional Cu(I) transfer from the low-affinity site (S2 site) on sCupA to the high-affinity site (S1 site) of CopAMBD. NMR solution structure of apo-sCupA adopts the same fold as the crystal structure of Cu(I)-bound sCupA with the exception of a flexible Cu(I) binding loop between $\beta$7 and $\beta$8. Backbone and side chain NMR dynamic study show the side chains of residues in S1 site are pre-arranged for Cu(I) binding while side chains of residues in S2 site are completely disordered. Cell growth analysis shows that the high affinity S1 Cu(I) site is dispensable for cellular Cu(I) resistance, while the low affinity S2 Cu(I) binding site is essential for bacterial growth under copper stress, which is in consistent with the hypothesis that S2 is more involved in Cu(I) transfer to CopA for efflux. All above establishes the first Cu(I) chaperone required for copper resistance in bacteria in contrast to the CopZ-like chaperones. The study in my dissertation leads to two further important questions which will require additional investigation. One is what the mechanism of a protein-protein interaction is between CupA and CopA and the second one is there any interplay between copper resistance and redox homeostasis systems in S. pneumoniae cells. The answers to those two questions may help us to better understand the biology of S. pneumoniae, which might shed light on combating the drug resistance issue of DRSP.