A Structural-Dynamical Characterization of Human Cox17

Molecular chaperone function of Mia40 triggers consecutive induced folding steps of the substrate in mitochondrial protein import

“…Once in the IMS, they must be trapped there by acquiring the mature, folded form. The following steps along the compartment-dependent folding process have been structurally characterized: the cytoplasmatic form of CHCH, CHCH-Mia40 covalent intermediate, the quasi-mature form CHCH 1S─S , and the mature CHCH 2S─S form (5). Side chains of the hydrophobic residues of Mia40 cleft, of the hydrophobic ITS residues of CHCH, and of cysteine residues are in red, blue, and yellow sticks, respectively; van der Waals contacts of the side chains constituting the hydrophobic cleft of Mia40 are shown as red dots surface.…”

“…hCox17-and hMia40-mutated proteins were expressed in E. coli BL21(DE3) gold cells following the same protocol reported for the wild-type proteins (5, 9). hCox17-and hMia40-mutated proteins were purified as previously described for the wild-type proteins (5,9). Wild-type and mutated yTim10 were similarly obtained Fig.…”

“…Cysteine to serine mutations of two or three of the four cysteines involved in the formation of the two structural disulfide bonds of human Cox17 (hCox17) or of yeast Tim10 (yTim10) as well as the cysteine to serine mutation of one of the two cysteines of the CPC motif of human Mia40 (hMia40) were generated by PCR-based site-directed mutagenesis from available pETG-30A, pDEST-MBP, and pGEX plasmids (5,9,27) containing, respectively, hMia40, hCox17, and yTim10 genes. hCox17-and hMia40-mutated proteins were expressed in E. coli BL21(DE3) gold cells following the same protocol reported for the wild-type proteins (5, 9).…”

“…Mitochondrial proteins sharing a coiled coil-helix-coiled coilhelix (CHCH) domain, like Cox17 and the small Tims, are reduced in the cytoplasm where they are released once synthesized, then they move to the IMS through the translocase of the outer membrane protein channel (2,3), and here they are trapped through a compartment-dependent oxidation of four cysteines that bridge the CHCH domain (4,5). Formation of disulfide bonds in vivo does not occur spontaneously but requires dedicated protein catalysts, which usually are part of an oxidative machinery able to introduce disulfide bonds in the final protein target (6).…”

Loop recognition and copper-mediated disulfide reduction underpin metal site assembly of Cu A in human cytochrome oxidase

“…All these proteins (including COX II) bind copper through two Cys ligands whose redox status is tightly linked to their function (19,20). Cox17 is a soluble metallochaperone that transfers Cu(I) ions to both Sco1 and Sco2 (21,22). Sco1 and Sco2 are homologous proteins, anchored to the inner mitochondrial membrane through a single transmembrane helix that locates their soluble, copper-binding domain in the IMS (15,23).…”