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. 2021 Sep 28;118(39):e2106950118.
doi: 10.1073/pnas.2106950118.

Molecular characterization of a complex of apoptosis-inducing factor 1 with cytochrome c oxidase of the mitochondrial respiratory chain

Johannes F Hevler  1   2 , Riccardo Zenezeni Chiozzi  1   2 , Alfredo Cabrera-Orefice  3 , Ulrich Brandt  3   4 , Susanne Arnold  5   4 , Albert J R Heck  6   2
Affiliations

Molecular characterization of a complex of apoptosis-inducing factor 1 with cytochrome c oxidase of the mitochondrial respiratory chain

Johannes F Hevler et al. Proc Natl Acad Sci U S A. .

Abstract

Combining mass spectrometry-based chemical cross-linking and complexome profiling, we analyzed the interactome of heart mitochondria. We focused on complexes of oxidative phosphorylation and found that dimeric apoptosis-inducing factor 1 (AIFM1) forms a defined complex with ∼10% of monomeric cytochrome c oxidase (COX) but hardly interacts with respiratory chain supercomplexes. Multiple AIFM1 intercross-links engaging six different COX subunits provided structural restraints to build a detailed atomic model of the COX-AIFM12 complex (PDBDEV_00000092). An application of two complementary proteomic approaches thus provided unexpected insight into the macromolecular organization of the mitochondrial complexome. Our structural model excludes direct electron transfer between AIFM1 and COX. Notably, however, the binding site of cytochrome c remains accessible, allowing formation of a ternary complex. The discovery of the previously overlooked COX-AIFM12 complex and clues provided by the structural model hint at potential roles of AIFM1 in oxidative phosphorylation biogenesis and in programmed cell death.

Keywords: AIFM1; COX; complexome profiling; cross-linking mass spectrometry; mitochondria.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Two-tier experimental strategy for the analysis of proteome-wide protein–protein interactions in BHM. Mitochondria membranes were cross-linked with either of the three cross-linking reagents, DSSO, PhoX, or DMTMM. Subsequently, samples were analyzed by XL-MS and complexome profiling. Identified cross-linked peptides were used to generate protein–protein interaction networks. Protein interactions and structural models of AIFM1 with COX were then computationally modeled using the distance restraints from XL-MS data together with the assembly state and stoichiometry information obtained by complexome profiling.
Fig. 2.
Fig. 2.
Dimeric AIFM1 forms a defined complex with monomeric COX. (A) Interaction network of AIFM1 in cross-linked BHM. Bold numbers indicate the observed cross-links for each interaction, and the thickness of lines indicate the cumulative evidence (CSMs) for each interaction (number in parentheses). Orange lines indicate cross-links involving AIFM1, while cross-links between AIFM1 interactors are presented as gray lines. (B) Xi-net plot of the COX-AIFM1 interaction. Purple colored links indicate intracross-links. Green colored links indicate intercross-links. Respective sequence and cross-link features are indicated accordingly. (C) Migration profiles of AIFM1 (orange) and averaged COX (green) from non–cross-linked (untreated) and cross-linked (PhoX or DMTMM) mitochondria separated by BN-PAGE (4 to 16%). Peaks are annotated based on the apparent molecular mass of AIFM1 (∼62 kDa) and monomeric COX (∼220 kDa). In all samples, peaks corresponding to monomeric AIFM1 and COX as well as a peak corresponding to a COX-AIFM12 complex are observed. Although already present in the non–cross-linked sample, treatment with DMTMM seems to somewhat stabilize the COX-AIFM12 complex.
Fig. 3.
Fig. 3.
Cross-link–derived structural model of the COX-AIFM12 complex. (A) Visualization of the cross-link–driven accessible interaction space models for a COX-AIFM12 complex. COX is represented in green, while the bright orange volume represents the center-of-mass position of the AIFM1 dimer, and the dark orange volume represents the center-of-mass position of the model of the AIFM1 N terminus (residues 55 to 124). The cross-linking data are consistent with the interaction space available for docking dimeric AIFM1 and the N-terminal region of one AIFM1 protomer to monomeric COX. (B) Cross-link–derived structural model of the COX-AIFM12 complex. COX is represented in green, and AIFM1 protomers (residues 128 to 516; 551 to 613) with and without N-terminal region (residues 55 to 127) are represented in orange and yellow, respectively. The transmembrane residues (67 to 85) of the N terminus of the interacting AIFM1 moiety is highlighted in red. Membrane boundaries of the inner mitochondrial membrane are sketched as gray spheres. The final complex consists of monomeric COX, dimeric AIFM1 (residues 128 to 516, 551 to 613), and the N-terminal region of one AIFM1 protomer (residues 55 to 127).
Fig. 4.
Fig. 4.
Deciphering interaction interfaces in the COX-AIFM12 structural model. (A) Three distinct interfaces between COX subunits and respective AIFM1 protomers were found. Subunits (COX) and protein domains (AIFM1) with residues in respective interfaces are colored in gray. Active COX residues are shown as green-colored sticks and active AIFM1 residues as orange-colored sticks. (B) Analysis of the number of residue contacts between respective COX subunits and AIFM1 domains. Colored circles indicate residue contacts between single subunits (COX) and domains (AIFM1) with the size of each circle corresponding to the number of residue–residue interactions. (C) COX-AIFM12 complex with CytC (purple) bound to its COX-binding site. The structural model presented here was merged with a previously published model of CytC docked to COX from bovine heart (54). COX subunits are colored green, while AIFM1 protomers are colored orange and yellow. The transmembrane (TM) domain of the N-terminal domain of AIFM1 is highlighted in red. Boundaries of the inner mitochondrial membrane are indicated as gray spheres.
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