Sco Proteins Have Distinctive Roles in the CuA Center Biogenesis

MARCOS MORGADA; LUCIANO ABRIATA; LUCIA BANCI; ALEJANDRO J. VILA

Chascomús

Congreso; Latin American Meeting in Biological and Bioinorganic Chemistry LABIC IV; 2014

INTRODUCTION The dinuclear copper center CuA is the electron entry point of the cytochrome c oxidase (COX). It funnels the electrons from reduced cytochrome c to the CuB center at subunit I of COX where O2 is reduced to water molecules. The correct assembly of this metal center is essential for the function of the complex and thus for the survival of the cell. Metallochaperones are proteins capable of binding copper ions and transfer them to their target proteins that use copper as a cofactor, and at the same time reducing the toxic effect that can be caused by free Cu(I) ions. Recently our group proposed a mechanism for the insertion of the copper ions to the CuA center of bacterial COX, following the copper transfer reaction using NMR. Finding that bacterial Sco main function, despite its ability to bind copper ions, is to keep the cysteines from the CuA center in the reduced state thus the copper ions can be transferred from a periplasmic metallochaperone (PCuAC) that act as a copper donor.1 In the case of humans, no homologue of PCuAC was found and biochemical studies shown that several proteins are involved in the assembly of the CuA center. Two of the identified proteins are Sco1 and Sco2. Mutations in either of these two proteins are lethal because of a deficiency in the assembly of the CuA center in the subunit II of COX (COX II).2 Structural studies showed that both proteins can act as metallochaperones because they are able to bind copper ions through a CxxxCxnH motif, and the presence of two Cys residues make them not only able to transfer copper but also able to act as thiol-disulphur oxidoreductase (Figure1).3,4  Until now the bottleneck to establish the role of each Sco in the assembly of the CuA center is the unavailability of the soluble domain of an eukaryotic COX II. Here we show, using a model of the eukaryotic oxidase (CuA*), that we have been able follow by NMR the interactions between these two putative metallochaperones finding that the role of Sco1 is to transfer the copper ions while Sco2 is important to maintain the Cys ligands in the reduced state.   EXPERIMENTAL METHODS Production of Protein Samples Unlabeled and uniformly labeled proteins were expressed in E. coli BL21-Gold (DE3) growing in either rich LB medium or M9 minimal medium supplemented with labeled or unlabeled ammonium chloride (1.0 g/L, 99% 15N when labeled). Purification of proteins from cell lysates was done as described elsewhere.3-5   Protein-Protein Interaction The interaction studies were performed in anaerobic conditions using 0.2-1.5 mM samples in 50mM potassium phosphate buffer pH 7 and followed by NMR. Holo-Sco1 or Holo-Sco2 samples were prepared as described elsewhere.3,4 The Cys redox state of the Sco?s and CuA* proteins, and of the protein mixtures was investigated by not-reducing SDS gel electrophoresis, after reaction with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, disodium salt (AMS) which alkylate free thiols adding a MW of ∼ 0. 5 kDa for each reduced cysteines. Reaction of the high molecular mass thiol-conjugating agent AMS molecule with a protein allows indeed separation of two bands in the SDS gel corresponding to the two protein redox states , i.e. with two reduced thiols or with a disulphide formed. Reduced and oxidized samples and protein mixtures were treated in anaerobic environment with 1% (w/v) SDS, 10 mM AMS for 1h at 37°C and resolved by 17% not-reducing SDS-PAGE.   RESULTS AND DISCUSSION The chimeric protein CuA* is able to form a CuA center by the addition of free copper ions.6 The 15N-1H HSQC spectra of the different conditions of CuA* (oxidized, reduced and metal bound) are very distinctive the making it suitable for the study of the copper transfer reactions using NMR. AMS test showed that Sco2 has more thiol-disulphur oxidoreductase activity in correlation with the reduction potential of each Sco and that this activity is enhanced by the binding of copper ion to the metal site. This is also supported by NMR because upon addition of Cu(I)-Sco2 to 15N labeled CuA* in its oxidized state (S-S)  the resulting 15N-1H HSQC spectrum corresponds to the reduced form of the protein (2SH), but is not able to transfer Cu ions efficiently since no metal transfer is observed. When Cu(I)-Sco1 is added to the mixture we observed the formation of the metal bound CuA* protein indicating that Sco is able to transfer copper ions to the reduced chimera. CONCLUSION The availability of the chimera and the very distinguishable 15N-1H HSQC of the three possible states of the protein (oxidized, reduced, metal bond) allowed us the use of NMR to follow the interactions in vitro between the putative metallochaperones and COX II. We found the principal role of each Sco protein in the assembly of the CuA site being Sco1 mainly involved in the transfer of the copper ions while Sco2 is principally involved in the reduction of the cysteines of COX II.   REFERENCES 1     Abriata, L. A. et al. Nat Chem Biol (2008) 4, 599-601. 2     Leary, S. C. et al. Hum.Mol.Genet. (2004) 13, 1839-1848. 3     Banci, L. et al. Structure (2007) 15, 1132-1140. 4     Banci, L. et al. Proc.Natl.Acad.Sci.U.S.A (2006) 103, 8595-8600. 5     Fernandez, C. O. et al. J.Am.Chem.Soc. (2001) 123, 11678-11685. 6     Morgada, M. N. et al. Angew Chem Int Ed Engl (2014), doi:10.1002/anie.201402083.