Molecular characterisation of a metallo-chaperone and a membrane copper pump in gram-negative bacteria: key proteins for copper tolerance and resistance

© 2017 Dr. Chathuri Jayanthika Kumari WijekoonCopper is an essential but potentially toxic element for all living organisms. It is required as a redox catalytic cofactor for enzymes involved in multiple processes. However, uncontrolled copper is potentially toxic due its redox activity and highly competitive binding affinities. Consequently, a balance between deficiency and excess must be maintained. Specific routes exist for the transport of copper within cells and errors in these pathways are associated with both acquired and inherited copper-based diseases. The internal pathways comprise mobile carrier proteins (metallo-chaperones) and membrane-bound transporters (pumps). Quantitative characterization of these proteins at molecular and atomic levels generally requires tens of milligrams of highly purified samples, a situation frequently challenged by problems in generating unmodified native forms. A variety of affinity tags, such as the popular poly-histidine tag, have been developed to facilitate purification but they generally rely on expensive affinity resins and their presence may interfere with protein characterization. This work documents that addition of a poly-lysine tag to the C-terminus of certain proteins enables ready purification in large scale via cost-effective cation-exchange chromatography. The tag may be removed readily by the enzyme carboxypeptidase B to generate the native protein with no extra residues. But this cleavage step is normally not necessary for most copper binding proteins since the poly-lysine tag is shown to have no detectable affinity for either Cu(I) or Cu(II) and imposes no interference to the copper binding properties of the target proteins. In contrast, the conventionally used poly-histidine tag possesses a sub-picomolar affinity for Cu(I) and nanomolar affinity for Cu(II) and may need to be removed for reliable characterization of the target proteins. The copper binding affinities of the poly-histidine tag has never been quantified before and these conclusions may be extended to the study of other metallo-proteins and metallo-enzymes. Under copper limiting conditions, the bacterium Pseudomonas fluorescens SBW25 activates the expression of two copper uptake proteins Pf-CopCD to import this essential nutrient. Pf-CopC is a periplasmic copper chaperone that may donate copper to the inner membrane transporter Pf-CopD for active copper importation. A database search revealed that Pf-CopC belongs to a new family of CopC proteins (designated Type B in this work) that differ significantly from the known CopC proteins of Type A that possess two separated binding sites specific for Cu(I) and Cu(II). This work reports the isolation and characterization of Pf-CopC and demonstrates that it lacks a Cu(I) binding site and possesses a novel Cu(II) site that binds Cu(II) with 100 times stronger affinity than do the Type A proteins. Presumably this is a requirement for a copper uptake role under copper limiting conditions. The Cu(II) site incorporates a highly conserved 'amino terminal copper and nickel' (ATCUN) binding motif NH2-Xxx-Xxx-His, but the anticipated ATCUN binding mode is prevented by a thermodynamically more favourable binding mode comprising His1 as a key bidentate ligand and His3 and His85 as co-ligands. However, upon His1 mutation, the ATCUN binding mode is adopted. This work demonstrates how a copper chaperone may fine-tune its copper-binding site to meet new challenges to its function. Cu-ATPases are selective for Cu(I) and are vital for activation of essential copper-dependent enzymes and for removal of excess copper from cell cytosols. Inactivation of the human copper pumps ATP7A and ATP7B is associated with the copper imbalance characteristic of Menkes and Wilson diseases and of a spectrum of neuro-pathologies. In the past decade, extensive in vivo cell studies have advanced the understanding of the cellular functions of Cu-ATPases. However, the mechanisms of binding and transport of Cu(I) and their coupling to ATP hydrolysis remain speculative. In particular, molecular study of purified samples, critical for comprehension of protein function, has lagged behind. The current challenges include the difficulty of expression and isolation of functional membrane enzymes and the lack of suitable assays for ATPase activity and copper transport. This work details quantitative approaches to address these challenges. In this work, the Cu-ATPase CopA from E. coli (EcCopA) was expressed and purified in fully functional form and demonstrated to bind Cu(I) with sub-femtomolar affinity. It was incorporated into the lipid membrane of giant unilamellar vesicles (GUVs) whose dimensions match those of eukaryotic cells. A quantitative ATPase activity assay was achieved via an 1H NMR approach for the enzyme dissolved either in detergent or embedded in GUV membrane. The ATPase activity varied with Cu(I) availability in an optimized assay solution for either enzyme form, demonstrating a quantitative correlation between ATPase activity and Cu(I) transport. Quantitative analysis of the Cu content trapped by the GUVs has led to the answer for a key question- How many Cu(I) ions does EcCopA transport per catalytic cycle? The answer is one