Archive | 2021
Structural and biochemical characterisation of the periplasmic copper chaperone AccA in Neisseria gonorrhoeae
Abstract
Neisseria gonorrhoeae, the causative agent for the disease gonorrhoea, colonises the human epithelia. N. gonorrhoeae utilizes an anaerobic respiration pathway during infection. By using a partial denitrification pathway, the gonococcus can reduce nitrite obtained from the host environment, facilitated by AniA, a Cu-containing nitrite reductase. A previous study has identified a Cu chaperone, AccA, required for AniA activity and it was found to contain 2 Cu sites.Cu-loaded AccA was purified, crystallised, and structurally resolved by X-ray diffraction to a resolution of 3.3 A. A structure of Cu-loaded AccA was generated and the binding ligands in the primary Cu-site were identified as H69, M80, H103, and M105. The secondary Cu-site was unable to be resolved. It was predicted that protein opens and closes around the primary Cu-site, and the secondary Cu-site was located in the unstructured C-terminal region. The binding affinity of AccA to Cu(I) was assayed using a competition assay. Removal of ligands identified in the crystal structure, as well as M107, showed reduced affinity for Cu(I). A mutant form (AccA-Ab) lacking all four identified binding residues was found to be unable to compete with the colorimetric ligands. A mutant form (AccA-Δ) lacking the C-terminal tail, was found to strongly bind Cu(I), but at a slower rate than WT-AccA.Continuous wave X-Band EPR identified the Cu(II) binding stoichiometry for AccA as ~1.6:1, while AccA-Ab showed ~0.5:1. AccA-Δ showed a 0.8:1 Cu(II) binding stoichiometry. Titration experiments indicate a preferential loading in AccA for the primary Cu-site. Desalting experiments indicated that Cu(II) bound more weakly to the secondary Cu-site. DEER EPR identified a maximum of 2 Cu ions per AccA molecule, which exist at an estimated 26 A apart. Direct Cu-loading of AniA from solution found roughly equal formation of Type I and Type II Cu-centres, and enzyme activity was observed by cyclic voltammetry. AniA loaded by WT-AccA also showed similar results. AccA-Ab and AccA-Δ were able to produce active AniA, but AccA-Ab loaded the sites in unequal amounts, while AccA-Δ showed preferential loading of the Type II Cu centre.