Effect of Cu(II) and Conserved Copper Binding Sites on Multicopper Oxidase CopA and Characterization of the BioMnOx

The microbial manganese removal process is believed to be the catalytic oxidation of Mn(II) by manganese oxidase. In this study, the multicopper oxidase CopA was purified and found to have high manganese oxidation activity in vitro and Cu(II) can significantly enhance its manganese oxidation activity. The gene site-directed mutagenesis was used to mutate four conserved copper binding sites of CopA and then obtain four mutant strains. The manganese removal efficiency of the four strains was determined to find that H120 is the catalytic active site of the CopA. Protein modification analysis of CopA obtained under different conditions by mass spectrometry revealed that the loss of Cu(Ⅱ) and the mutation of the conserved copper binding site H120 resulted in the loss of modification of ethoxyformyl and quinone, the number of modifications was reduced and the position of modification was changed, eventually causing a decrease in protein activity. It reveals that Cu(II) and H120 play an indispensable role in the manganese oxidation of the multicopper oxidase CopA. The Mn valence state of BioMnOx was analyzed by XPS, finding that both the strain-mediated product and the CopA-mediated product were composed of MnO2 and Mn3O4 and the average valence of Mn is 3.2.

Mutation of copper binding sites on cellular prion protein abolishes its inhibitory action on NMDA receptors in mouse hippocampal neurons

We have previously reported that cellular prion protein (PrPC) can down-regulate NMDA receptor activity and in a copper dependent manner. Here, we employed AAV9 to introduce murine cellular prion protein into mouse hippocampal neurons in primary cultures from PrP null mice to determine the role of the six copper binding motifs located within the N-terminal domain of PrPC. The results demonstrate that viral expression of wild type PrPC lowers NMDAR activity in PrP null mouse hippocampal neurons by reducing the magnitude of non-desensitizing currents. Elimination of the last two copper binding sites alone, or in combination with the remaining four attenuates this protective effect. Thus our data suggest that copper ion interactions with specific binding sites on PrPC are critical for PrPC dependent modulation of NMDA receptor function.

Combination of Copper Ions and Nucleotide Generates Aggregates from Prion Protein Fragments in the N-Terminal Domain

Background: It has been previously found that PrP23-98, which contains four highly conserved octarepeats (residues 60-91) and one partial repeat (residues 92-96), polymerizes into amyloid-like and proteinase K-resistant spherical aggregates in the presence of NADPH plus copper ions. Objective: We aimed to determine the requirements for the formation of these aggregates. Methods: In this study, we performed an aggregation experiment using N-acetylated and Camidated PrP fragments of the N-terminal domain, Octa1, Octa2, Octa3, Octa4, PrP84−114, and PrP76−114, in the presence of NADPH with copper ions, and focused on the effect of the number of copper-binding sites on aggregation. Results: Among these PrP fragments, Octa4, containing four copper-binding sites, was particularly effective in forming aggregates. We also tested the effect of other pyridine nucleotides and adenine nucleotides on the aggregation of Octa4. ATP was equally effective, but NADH, NADP, ADP, and AMP had no effect. Conclusion: The phosphate group on the adenine-linked ribose moiety of adenine nucleotides and pyridine nucleotides is presumed to be essential for the observed effect on aggregation. Efficient aggregation requires the presence of the four octarepeats. These insights may be helpful in the eventual development of therapeutic agents against prion-related disorders.

Homology- and coevolution-consistent structural models of bacterial copper-tolerance protein CopM support a ‘metal sponge’ function and suggest regions for metal-dependent protein-protein interactions

Copper is essential for life but toxic, therefore all organisms control tightly its intracellular abundance. Bacteria have indeed whole operons devoted to copper resistance, with genes that code for efflux pumps, oxidases, etc. Recently, the CopM protein of the CopMRS operon was described as a novel important element for copper tolerance in Synechocystis. This protein consists of a domain of unknown function, and was proposed to act as a periplasmic/extracellular copper binder. This work describes a bioinformatic study of CopM including structural models based on homology modeling and on residue coevolution, to help expand on its recent biochemical characterization. The protein is predicted to be periplasmic but membrane-anchored, not secreted. Two disordered regions are predicted, both possibly involved in protein-protein interactions. The 3D models disclose a 4-helix bundle with several potential copper-binding sites, most of them largely buried inside the bundle lumen. Some of the predicted copper-binding sites involve residues from the disordered regions, suggesting they could gain structure upon copper binding and thus possibly modulate the interactions they mediate. All models are provided as PDB files in the Supporting Information and can be visualized online at http://lucianoabriata.altervista.org/modelshome.htmlNote (January 2017): Recent X-ray structures of apo, copper- and silver-bound CopM are < 3Å RMSD away from the models, and reveal metal-dependent structural flexibility (Zhao et al Acta Crystallogr D Struct Biol. 2016)