MIA40 circumvents the folding constraints imposed by TRIAP1 function

Abstract

Folding intermediates; Molten globule; Oxidative folding

Intermediarios de plegamiento; Glóbulo fundido; Plegamiento oxidativo

Intermedis de plegament; Glòbul fos; Plegament oxidatiu

The MIA40 relay system mediates the import of small cysteine-rich proteins into the intermembrane mitochondrial space (IMS). MIA40 substrates are synthesized in the cytosol and assumed to be disordered in their reduced state in this compartment. As they cross the outer mitochondrial membrane, MIA40 promotes the oxidation of critical native disulfides to facilitate folding, trapping functional species in the IMS. Here, we study the redox-controled folding of TRIAP1, a small cysteine-rich protein with moonlighting function: regulating phospholipid trafficking between mitochondrial membranes in the IMS and preventing apoptosis in the cytosol. TRIAP1 dysregulation is connected to oncogenesis. Although TRIAP1 contains a canonical twin CX9C motif, its sequence characteristics and folding pathway deviate from typical MIA40 substrates. In its reduced state, TRIAP1 rapidly populates a hydrophobic collapsed, alpha-helical, and marginally stable molten globule. This intermediate biases oxidative folding towards a non-native Cys37-Cys47 kinetic trap, slowing the reaction. MIA40 accelerates TRIAP1 folding rate by 30-fold, bypassing the formation of this folding trap. MIA40 drives the oxidation of the inner disulfide bond Cys18-Cys37, and subsequently, it can catalyze the formation of the outer disulfide bond Cys8-Cys47 to attain the native two-disulfide-bridged structure. We demonstrate that, unlike most MIA40 substrates, TRIAP1's folding pathway is strongly constrained by the structural requirements for its function in phospholipid traffic at the IMS. The obligatory population of a reduced, alpha-helical, metastable molten globule in the cytoplasm may explain TRIAP1's connection to the p53-dependent cell survival pathway, constituting a remarkable example of a functional molten globule state.

This work was funded by the Spanish Ministry of Science and Innovation (MICINN, Spain) (PID2022-137963OB-I00), by ICREA (ICREA-Academia 2020, Spain), and by CERCA Programme (Generalitat de Catalunya) to S. V. J. P., M. G.-G., and M. F.-S. were supported by the Spanish Ministry of Science and Innovation via a doctoral grant (FPU14/07161, FPU16/02465, and FPU20/02897), respectively.