Anna Janicka

A Structural-Dynamical Characterization of Human Cox17

Human Cox17 is a key mitochondrial copper chaperone responsible for supplying copper ions, through the assistance of Sco1, Sco2, and Cox11, to cytochrome c oxidase, the terminal enzyme of the mitochondrial energy transducing respiratory chain. A structural and dynamical characterization of human Cox17 in its various functional metallated and redox states is presented here. The NMR solution structure of the partially oxidized Cox17 (Cox172S-S) consists of a coiled coil-helix-coiled coil-helix domain stabilized by two disulfide bonds involving Cys25-Cys54 and Cys35-Cys44, preceded by a flexible and completely unstructured N-terminal tail. In human Cu(I)Cox172S-S the copper(I) ion is coordinated by the sulfurs of Cys22 and Cys23, and this is the first example of a Cys-Cys binding motif in copper proteins. Copper(I) binding as well as the formation of a third disulfide involving Cys22 and Cys23 cause structural and dynamical changes only restricted to the metal-binding region. Redox properties of the disulfides of human Cox17, here investigated, strongly support the current hypothesis that the unstructured fully reduced Cox17 protein is present in the cytoplasm and enters the intermembrane space (IMS) where is then oxidized by Mia40 to Cox172S-S, thus becoming partially structured and trapped into the IMS. Cox172S-S is the functional species in the IMS, it can bind only one copper(I) ion and is then ready to enter the pathway of copper delivery to cytochrome c oxidase. The copper(I) form of Cox172S-S has features specific for copper chaperones.

Identification of a novel high affinity copper binding site in the APP(145–155) fragment of amyloid precursor protein

The copper(II) binding features of the APP(145-155) and APP(145-157) fragments of the amyloid precursor protein, Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-NH2 and Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-Glu-Thr-NH2 were studied by NMR spectroscopy and NMR findings were supported by UV-vis, CD and EPR spectra. Potentiometric measurements were performed only for the more soluble Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-Glu-Thr-NH2 peptide fragment. The following was shown: (i) the imidazole rings of all the three His residues are involved in metal coordination; (ii) metal binding induces ionisation of Leu-148 and His-149 amide nitrogens that complete the donor set to copper(II) in the species dominant at neutral pH; (iii) the unusual coordination scheme of the His-Xxx-His-Xxx-His consensus sequence justifies the high specificity for Cu(II) when compared to SOD-like or albumin-like peptides or even in amyloid Abeta fragments. The present findings may represent the key for interpreting the observed requirement of His residues conservation for the redox cycling between Cu(II) and Cu(I) by soluble APP.

Copper-ion interaction with the 106–113 domain of the prion protein: a solution-equilibria study on model peptides

Prion diseases are characterized by a structural modification of the regular prion protein (PrP(C)) to its isoform, termed PrP(Sc)(scrapie). Such a modification involves the secondary and tertiary structure of the protein; the amino acidic sequence remains unchanged. PrP(Sc) is almost insoluble in non-denaturing solvents, resistant to proteases and it loses its redox activity. PrP(C) is able to bind copper and other metal ions: these complexes have been suggested to play an important role in the protein refolding leading to PrP(Sc). It is well-known that at least one relatively strong copper-binding site is located in the PrP(92--126) domain, where two His residues (96 and 111) are present. However, in the same domain, other amino acidic residues bear potentially donating atoms, i.e. Met, Asn and Lys residues. In order to shed light on the role of the side chains of such potentially tridentate amino acids on copper complexation, the polypeptide Ac-KTNMKHMA-NH(2), corresponding to the PrP(106--113) fragment, and some synthetic analogues have been investigated as ligands for the copper ion, by means of both thermodynamic and spectroscopic techniques. The pivotal role of imidazolic side chain of His in anchoring the metal ion has been confirmed. On the other hand, no clue was found on the participation of sulfur atom of Met or side amino-group of Lys residues to copper complex-formation.