Cryo-EM structure of human mitochondrial trifunctional protein

Abstract

The mitochondrial trifunctional protein (TFP) catalyzes three reactions in the fatty acid β-oxidation process. Mutations in the two TFP subunits cause mitochondrial trifunctional protein deficiency and acute fatty liver of pregnancy that can lead to death. Here we report a 4.2-Å cryo-electron microscopy α2β2 tetrameric structure of the human TFP. The tetramer has a V-shaped architecture that displays a distinct assembly compared with the bacterial TFPs. A concave surface of the TFP tetramer interacts with the detergent molecules in the structure, suggesting that this region is involved in associating with the membrane. Deletion of a helical hairpin in TFPβ decreases its binding to the liposomes in vitro and reduces its membrane targeting in cells. Our results provide the structural basis for TFP function and have important implications for fatty acid oxidation related diseases.

Keywords: cryo-EM; fatty acid β-oxidation; mitochondrial trifunctional protein.

Fig. 1.

Cryo-EM structure of the human TFP complex. (A) Schematic diagram of the mitochondrial fatty acid β-oxidation pathway. MOM, mitochondrial outer membrane; MIM, mitochondrial inner membrane; OXPHOS, oxidative phosphorylation. (B) The cryo-EM density map of the human TFP complex. The two TFPα subunits are shown in magenta and orange, and the two TFPβ subunits are shown in cyan and green.

Fig. 2.

Different quaternary structures of human TFP, pfTFP, and mtTFP. (A) The human TFP tetramer shown in two orientations. The insertion region in the ECH domain of TFPα is colored blue; and the two insertions in TFPβ are colored yellow and red. The CH2A–CH2B helical hairpin and the H9A helix are indicated. The active sites of TFPα (ECH: Glu151, Glu173; HACD: His498, Glu510) and TFPβ (Cys138, His428, Cys458) are indicated with black circles. The directions of how the substrate is presumably transferred between the three active sites are illustrated with arrows. (B) The structure of pfTFP [Protein Data Bank (PDB) ID code 1WDK] shown in the same orientations and color schemes as the human TFP in A. (C) The structure of mtTFP (PDB ID code 4B3H).

Fig. 3.

TFPβ_HH is involved in the membrane association of TFPβ. (A) The cryo-EM density map before the detergent-free mask is applied, with the structure model docked into the map. The TFPβ_HH regions in TFPβ are highlighted with a blue rectangle. The noncontinuous density shown in the red oval below the TFP tetramer presumably corresponds to the detergent molecules. (B) TFPβ_HH is important for the liposome binding of TFPβ. Cardiolipin-containing liposomes were mixed with the indicated proteins and incubated for 1 h, before the addition of Optiprep reagent to a final concentration of 35%. After centrifugation, 200-μL aliquots were taken out from different layers, from top to bottom, and analyzed by Western blot using an anti-TFPα antibody and an anti-His antibody that recognizes the His tag on TFPβ. Liposomes are enriched in the top layers after centrifugation. (The TFPα in the WT and mutant TFP complexes are untagged and therefore appear smaller on the gel compared with the His-tagged TFPα.) (C) TFPβ_HH is important for the membrane association of TFPβ in cells. C-terminally V5-tagged TFPα and C-terminally Flag-tagged TFPβ were expressed in HEK293A cells as indicated. The mitochondria of these cells were isolated, and the soluble proteins were separated from the mitochondria membranes by sonication and ultracentrifugation. COX2 is an inner mitochondrial membrane protein, and LRP130 is a mitochondrial matrix protein. Mito, mitochondria; mem, membrane.

Fig. 4.

TFP residues mutated in patients. (A) The TFP structure is shown as white ribbons. The Cα atoms of the TFP residues that are mutated in patients are shown as red spheres. The missense mutations found in TFPα include L130P, G148R, Q186E, R235W, A244V, V282D, I305N, L342P, V412L, A478P, A478V, E510Q, V599M, R676C, R676H, R676L, C688Y, D701G, and L733P. The missense mutations found in TFPβ include G59D, R61C, R61H, T62A, N114D, N114S, R117G, L121P, T133P, N142K, R229L, D242G, R247C, R247H, D263G, G280D, P294L, P294R, G301S, G301D, N307D, A326P, H379R, N389D, R444K, V455G, and A459E. Among them, TFPα-E510Q is the most common mutant. (B) Arg235 in TFPα appears to make hydrogen bonds with the LA2 helix in TFPβ. The electron density map is shown as light blue meshes. (C) Arg676 in TFPα appears to make a hydrogen bond with the main chain carbonyl group of Arg732. (D) Arg61 in TFPβ likely interacts with Phe64 and Asp263.