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

Abstract

Several proteins of the mitochondrial intermembrane space are targeted by internal targeting signals. A class of such proteins with α-helical hairpin structure bridged by two intramolecular disulfides is trapped by a Mia40-dependent oxidative process. Here, we describe the oxidative folding mechanism underpinning this process by an exhaustive structural characterization of the protein in all stages and as a complex with Mia40. Two consecutive induced folding steps are at the basis of the protein-trapping process. In the first one, Mia40 functions as a molecular chaperone assisting α-helical folding of the internal targeting signal of the substrate. Subsequently, in a Mia40-independent manner, folding of the second substrate helix is induced by the folded targeting signal functioning as a folding scaffold. The Mia40-induced folding pathway provides a proof of principle for the general concept that internal targeting signals may operate as a folding nucleus upon compartment-specific activation.

Fig. 1.

¹H-¹⁵N HSQC spectra of apoCox17. (A) Fully reduced (Cox17_6SH); (B) in-cell Cox17; (C) oxidized with two disulfide bonds (Cox17_2S─S).

Fig. 2.

Structural characterization of substrate-Mia40 covalent adducts. (A) The solution structure of the Cox17–Mia40 complex: The Mia40-induced α-helix in Cox17 is in green, the NHs chemical shift variations of Mia40 residues upon complex formation are mapped in red, and the intermolecular disulfide bond is in yellow. (B) Hydrophobic residues involved in the protein–protein recognition between Cox17 (in green) and Mia40 (in cyano) are shown in blue and red, respectively. Inter- and intramolecular disulfide bonds are in yellow; van der Waals contacts are shown in blue and red dots. (C) Experimental data-driven docking model of Tim9 peptide–Mia40 complex: The Mia40-induced α-helix in Tim9 peptide is in green, the NHs chemical shift variations of Mia40 residues upon complex formation are mapped in red, and the intermolecular disulfide bond is in yellow. (D) Hydrophobic residues involved in the protein–protein recognition between Tim9 peptide (in green) and Mia40 (in cyano) are shown in blue and red, respectively. Inter- and intramolecular disulfide bonds are also shown in yellow; van der Waals contacts are shown in blue and red dots.

Fig. 3.

Compartment-dependent folding mechanism of CHCH proteins. Unfolded CHCH proteins enter mitochondria via a translocase of the outer membrane (TOM) channel. To do so, they have conformational plasticity in order both to sneak in and to optimize the interactions with TOM. 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.