The Mysterious Multitude: Structural Perspective on the Accessory Subunits of Respiratory Complex I

Abstract

Complex I (CI) is the largest protein complex in the mitochondrial oxidative phosphorylation electron transport chain of the inner mitochondrial membrane and plays a key role in the transport of electrons from reduced substrates to molecular oxygen. CI is composed of 14 core subunits that are conserved across species and an increasing number of accessory subunits from bacteria to mammals. The fact that adding accessory subunits incurs costs of protein production and import suggests that these subunits play important physiological roles. Accordingly, knockout studies have demonstrated that accessory subunits are essential for CI assembly and function. Furthermore, clinical studies have shown that amino acid substitutions in accessory subunits lead to several debilitating and fatal CI deficiencies. Nevertheless, the specific roles of CI's accessory subunits have remained mysterious. In this review, we explore the possible roles of each of mammalian CI's 31 accessory subunits by integrating recent high-resolution CI structures with knockout, assembly, and clinical studies. Thus, we develop a framework of experimentally testable hypotheses for the function of the accessory subunits. We believe that this framework will provide inroads towards the complete understanding of mitochondrial CI physiology and help to develop strategies for the treatment of CI deficiencies.

Keywords: accessory subunits; electron transport chain; mitochondrial complex I; mitochondrial diseases; oxidative phosphorylation (OXPHOS).

FIGURE 1

Structure of mammalian CI from O. aries (PDB: 6ZKC). (A) The 14 core subunits are shown as colored surfaces. The accessory subunits are shown as grey cartoons. The CI boot-shape analogy is indicated at the top of the panel. (B) The 31 accessory subunits are shown as colored surfaces while the core subunits are shown as grey cartoons.

FIGURE 2

Mammalian CI Assembly. (A) The modular assembly pathway of mammalian mitochondrial CI as proposed by Guerrero-Castillo et al. (2017a). The structures of CI subunits within each subassembly are shown as atomic spheres and colored by subassembly. The subunits constituting each subassembly are indicated in shorthand notation (e.g., NDUFS1 is shortened to S1). The assembly factors found associated with each subassembly are indicated in purple. Subunits that associate with intermediate assemblies (e.g., the Q/P_P subassembly) are shown in beige. The terminally associated subunits are shown in magenta. (B) CI structure shown as atomic spheres with subunits colored by their subassembly as in panel (A). Subunits that associate with intermediate assemblies independent of the modules are shown in beige and terminally associated subunits are shown in magenta.

FIGURE 3

Ancient accessory subunits of CI. (A) NDUFS4 (berry), (B) NDUFS6 (plum) and (C) NDUFA12 (gold) are shown as colored surfaces on CI, the core subunits are shown as colored cartoons and other accessory subunits are shown as grey cartoons. Shin view of (D) mammalian (O. aries PDB: 6ZKC throughout), (E) E. coli (PDB: 7NZ1 throughout) and (F) T. thermophilus (PDB: 4HEA throughout) CI structures shown as transparent surfaces. NDUFS4 and the NDUFS1 loops (orange) that differ between the structures are shown as cartoons. (G) Calf view of Y. lipolytica CI structure (PDB ID: 6YJ4) shown as transparent surfaces with NDUFS6 (NUMM) and NDUFA12 (N7BM) shown as cartoons with the Zn²⁺ ion shown as atomic sphere and coordinating residues shown as sticks colored by element. Calf view of (H) mammalian, (I) E. coli and (J) T. thermophilus CI structures shown as transparent surfaces with NDUFS6 and the NDUFS8 (sea green) loops that differ between the structures labeled and shown as cartoons. The FeS clusters and Zn²⁺ ions are shown as atomic spheres and colored by element. (K) Structural differences between assembly factor NDUFAF2 and accessory subunit NDUFA12. Calf view of CI showing assembly factor NDUFAF2 from Y. lipolytica (PDB: 6RFQ) superposed on NDUFA12 (N7BM) (PDB: 6RFR). The transparent surface of CI is shown with NDUFA12 as cartoon in gold and NDUFAF2 shown as cartoon colored in dark olive. (L) Lipid binding around the Q-tunnel. Looking up from the Y. lipolytica (PDB: 6YJ4) CI heel showing the accessory subunits NDUFA9 (NUEM) (lavender), NDUFA12 (N7BM) and core subunit NDUFS7 (NUKM) (tea green) as cartoon. Other subunits are shown as transparent surfaces. Lipids are shown as spheres colored by element. (M) The interface between NDUFS4 (NUYM), NDUFA12 (N7BM) and NDUFS6 (NUMM) of Y. lipolytica CI (PDB: 6RFR). NDUFS4, NDUFA12 and NDUFS6 are shown as surfaces and all other CI subunits are shown as cartoon. (N) Clash between NDUFS6 and NDUFAF2. Y. lipolytica NDUFAF2 (PDB: 6RFQ) superposed on to NDUFA12 (N7BM) (PDB: 6RFR). NDUFS6 (NUMM) and NDUFAF2 are shown as cartoon. NDUFA12 (N7BM) is not shown for clarity. (O) Schematic diagram showing the role of ancient accessory subunits in the “checkpoint” hypothesis of the CI assembly pathway. NDUFS4 binds to CI Q/P subassembly bound to NDUFAF2 and disengages the C-terminal unstructured region of NDUFAF2. Addition of NDUFS6 and N-module to the subassembly results in the release of NDUFAF2 and frees up the NDUFA12 binding site. NDUFA12 binds to the subassembly at the site of NDUFAF2 and completes the assembly. Subunits are colored as in Figure 1 throughout unless stated otherwise. NDUFS4: berry, NDUFS6: plum, NDUFA12: gold, NDUFS1: orange, NDUFS8: sea green, NDUFAF2: dark olive, NDUFA9: lavender, NDUFS7: tea green.

FIGURE 4

Accessory subunits NDUFV3 and NDUFA2. NDUFV3-S (short isoform) (purple) (A), NDUFV3-L (long isoform) (purple) (B) and NDUFA2 (blue) (C) are shown as colored surface on CI (PDB: 6ZKC). Disordered N-terminal of the (A) short isoform (64 aa) and (B) long isoforms (423 aa) are represented by the purple line. Red square in (B) represents the conserved string of serine residues. The core subunits are shown as cartoons colored as in Figure 1A and the other accessory subunits are shown as grey cartoon. (D) The mammalian reduced NDUFA2 structure shown as cartoon. The cysteines are shown in stick representation and colored by element (PDB: 6ZKC). (E) NMR solution structure of oxidized human NDUFA2 (PDB: 1S3A) superposed onto reduced NDUFA2 found in mammalian CI (PDB: 6ZKC). Reduced NDUFA2 is shown in blue cartoon and the oxidized NDUFA2 is shown in red cartoon. (F) NMR solution structure of human NDUFA2 (PDB: 1S3A) superposed onto reduced NDUFA2 seen in mammalian CI. The clash caused due to rearrangement of loops [β1-α1^A2 (aa 23–29) and β2-3^A2 (aa 57–63)] is shown by the star. (G-I) NDUFA2 compensates for the loss of NDUFS1 domain D. Domain architecture of NDUFS1 (G) mammalian (O. aries) (PDB:5LNK), (H) E. coli (PDB: 7NZ1) (I) T. thermophilus (PDB:4HEA) is shown as surfaces colored and labeled according to the domains. (J) Schematic representation of the partial dissociation of CI due to oxidation of cysteines in NDUFA2. ROS generated at the FMN site oxidizes the cysteines in NDUFA2 resulting in the formation of a disulfide bond. The rearranged NDUFA2 loop clashes with NDUFS1 resulting in the dissociation of NDUFA2. The dissociation of NDUFA2 destabilizes NDUFS1 and the N-module. This destabilization eventually results in the N-module dissociating. Subunits are colored as in Figure 1 in all the structure figures unless stated otherwise. NDUFV3: purple, reduced NDUFA2: blue, oxidized NDUFA2: red.

FIGURE 5

Accessory subunits NDUFA5 and NDUFA7. (A) The structure of CI (PDB: 6ZKC) in cartoon with the surface of NDUFA5 (auburn) shown, the core subunits colored as in Figure 1A and the accessory subunits grey. (B–D) The NDUFA5 C-terminus compensates for a shorter NDUFS3 (corn yellow) β3-4^S3 loop. (B) Mammalian (PDB: 6ZKC), (C) T. thermophilus (PDB: 4HEA) and (D) E. coli (PDB: 7NZ1). CI structures shown in transparent surface with NDUFA5 and the NDUFS3 β3-4^S3 loop in cartoon. (E–G) NDUFA5 may compensate for a shorter NDUFS3 β4-5^S3 loop. (E) O. aries cartoon of NDUFS3 β4-5^S3 loop (aa 99–102), NDUFA5, and NDUFS8 (sea green) on the CI surface (PDB: 6ZKC). The β4-5^S3 loop is shorter in O. aries than in bacteria. (F) T. thermophilus cartoon of the β4-5^S3 loop (aa 76–86) and NDUFS8 (Nqo9) on the CI surface (PDB: 4HEA). (G) Y. lipolytica cartoon of the NDUFS3 β4-5^S3 (NUGM) loop (aa 147–150), NDUFA5 (NUFM) and NDUFS8 (NUPM) on the CI surface (PDB: 6YJ4). Y. lipolytica has an extended NDUFS8 coil compared to O. aries and T. thermophilus. (H) The structure of CI (PDB: 6ZKC) shown in cartoon with the surface of NDUFA7 (azzure blue), the core subunits colored as in Figure 1 and the accessory subunits grey. (I–K) Differences in NDUFA7 between eukaryotes. (I) O. aries NDUFA7 shown as cartoon on the CI (PDB: 6ZKC) surface with the variable region indicated (*) and the N-terminal coil and helix of NDUFS3 shown as cartoon. A: ascending coil, L: lateral coil, D: descending coil. (J) Y. lipolytica NDUFA7 (NUZM) shown as cartoon on the CI (PDB: 4HEA) surface with the variable region indicated (*) and the N-terminal coil and helix and C-terminal coil of NDUFS3 (NUGM) shown as cartoon. (K) V. radiata NDUFA7 (NDUA7) shown as cartoon on the CI (PDB: 6X89) surface with the variable region indicated (*) and the N-terminal coil and helix of NDUFS3 (NDUS3) shown as cartoon. (L) A conformation change is needed in the N-terminal helix of NDUFS3 relative to that from T. thermophilus to accommodate NDUFA7. Cartoon of NDUFS3 in O. aries (PDB: 6ZKC) and NDUFS3 (Nqo5; grey) in T. thermophilus (PDB: 4HEA) on the O. aries CI surface. (M,N) NDUFA7 participates in NDUFA12 (gold) binding. (M) Cartoon of NDUFA7 (NUZM), the N-terminal and C-terminal loop of NDUFS3 (NUGM) and NDUFA12 (N7BM) on the CI surface (PDB: 6YJ4). (N) Cartoon of NDUFA7, NDUFS3 and NDUFAF2 (N7BML) (gold) on the CI surface (PDB: 6RFQ). The N-terminal regions of NDUFA7 and NDUFS3 are disordered. (O–Q) Stabilizing role for NDUFA7 at the interface of the N and Q modules. (O) O. aries cartoon of NDUFA7 on CI surface (PDB:6ZKC). NDUFA7 stabilizing interactions are replaced by core subunits in bacteria. A: ascending coil, L: lateral coil. (P) T. thermophilus cartoon of NDUFS1 (orange) (Nqo1) on CI surface (PDB: 4HEA). (Q) E. coli cartoon of C-terminal coil of NDUFV1 (NuoF) on CI surface (PDB: 7NZ1). Subunits are colored as in Figure 1 throughout unless stated otherwise. NDUFA5: auburn, NDUFS3: corn yellow, NDUFS8: sea green, NDUFA7: azzure blue, NDUFA12: gold, NDUFS1: orange.

FIGURE 6

Accessory subunits NDUFA6, NDUFAB1-α, NDUFB9, and NDUFAB1-β in mammalian CI. (A) The structure of CI in cartoon with the surfaces of NDUFA6 (cinnabar red)/NDUFAB1-α (violet) and NDUFB9 (yellow orange)/NDUFAB1-β (violet) shown, the core subunits colored as in Figure 1A and the accessory subunits grey. (B) Shin view CI showing the NDUFA6/NDUFAB1-β pair in cartoon and other CI subunits as transparent surfaces. The acylated 4′-phosphopantetheine group is shown in spheres (PDB: 5LNK). (C) Looking down on the toe of CI (PDB: 6ZKC) showing the NDUFB9/NDUFAB1-β pair in cartoon and the other CI subunits as transparent surfaces. (D–F) NDUFA6 replaces structural elements of bacterial CI. Shin view of (D) mammalian (PDB: 5LNK), (E) E. coli (PDB: 7NYR) and (F) T. thermophilus CI shown as transparent surface with NDUFA6, the NDUFS2/S3 (NuoCD) (corn yellow) linker and NDUFS3 C-termini shown in cartoon. Accessory subunits that are not important for the discussion are omitted from panel (D) for clarity. The NDUFS2/S3 (NuoCD) linker in E. coli and Nqo16 (pink) subunit in T. thermophilus conflict with the position of NDUFA6 in mammals. NDUFA6: cinnabar red, NDUFAB1-α: violet, NDUFB9: yellow orange, NDUFAB1-β: violet/violet, NDUFS2/S3: corn yellow, Nqo16: pink.

FIGURE 7

Accessory subunit NDUFA9. (A) The structure of CI (PDB: 6ZKC) in cartoon with the surface of NDUFA9 (lavender) shown, the core subunits colored as in Figure 1A and the accessory subunits grey. (B) M. musculus CI (PDB: 6G2J) and (C) Y. lipolytica CI (PDB: 6RFR) shown as transparent surfaces with NDUFA9 (NUEM), ND3 (sea green) and ND6 (cyan-blue) shown in cartoon. NADPH and lipids are shown as spheres. The C-terminal coils of NDUFA9 are colored red. The key residues interacting with ND6 are shown in stick representation. Accessory subunits that are not important for the discussion are omitted from the figures for clarity. Subunits are colored as in Figure 1 in all the structure figures unless stated otherwise. NDUFA9: lavender, ND3: sea green, ND6: cyan-blue.

FIGURE 8

Accessory subunits of the CI heel. The structure of CI (PDB: 6ZKC used throughout) in cartoon with the surface of NDUFA13 (light yellow) (A), NDUFA3 (pine green) (B) and NDUFA1 (firebrick red) (C) shown, the core subunits colored as in Figure 1A and the accessory subunits grey. (D) Looking up from the CI heel showing NDUFA13, NDUFA3, and NDUFA1 of H. sapiens CI structure (PDB: 5XTD) as cartoons. NDUFA8 (lilac purple) is shown as a transparent surface for clarity. (E) Accessory subunit NDUFA13 of H. sapiens CI structure (PDB: 5XTD) is shown as cartoon. The residues mentioned in the text are shown in stick representation. (F) NDUFA13 compensates for the lost transmembrane helix TMH9^ND1 (cobalt blue) of T. thermophilus. T. thermophilus ND1 (Nqo8) (PDB: 4HEA used throughout) is superposed on ND1 of mammalian (O. aries) CI structure shown as transparent surface. TMH5^ND1(Nqo8) and TMH9 ^ND1(Nqo8) are shown as cartoons. NDUFA13 surface and cartoon are shown. (G) Schematic representation of the “Flag post model.” NDUFA13 of healthy CI is furled and incapable of binding to STAT3. NDUFA13 alone is raised exposing the STAT3 interacting site to the IMS. (H,I) Assembly vs. lipid binding roles of NDUFA3. (H) Mammalian CI shown as transparent surface with NDUFA3, NDUFA13, α1^ND1 (cobalt blue), and α3^ND3 (sea green) shown as cartoons. (I) Y. lipolytica CI (PDB: 6YJ4) shown as transparent surface with NDUFA3 (NI9M), NDUFA13 (NB6M), NUXM (light cyan), α1^ND1 (NU1M), and α3^ND3 (NU3M) (sea green) shown as cartoons. (J,K) NDUFA1 compensates for the shorter TMH2-3^ND1 loop. (J) Mammalian CI structure shown as transparent surface with NDUFA1, TMH1^ND1, TMH7^ND1 and the TMH2-3^ND1 loop shown as cartoons. (K) T. thermophilus CI shown as transparent surface with TMH1^ND1(Nqo8), TMH7^ND1(Nqo8) and the TMH2-3^ND1(Nqo8) loop shown as cartoon. (L) NDUFA1 connects the P_P-a and P_P-b subassemblies. Mammalian CI a transparent surface with NDUFA1 and the ND6 TMH4-5^ND6 (cyan-blue) loop shown as cartoon. Residues mentioned in the text are shown as sticks. Key H40-S46 sequence shown in purple. Subunits are colored as in Figure 1 in all panels unless stated otherwise. NDUFA13: light yellow, NDUFA3: pine, NDUFA1: firebrick red, NDUFA8: lilac purple, ND1: cobalt blue, ND3: sea green, ND6: cyan-blue, NUXM (light cyan).

FIGURE 9

Accessory subunit NDUFA10. (A) The structure of CI (PDB: 6ZKC) in cartoon with the surface of NDUFA10 (cadet blue) shown, the core subunits colored as in Figure 1A and the accessory subunits grey. (B) Transparent surface and cartoon of O. aries NDUFA10 (PDB: 6ZKC). The P-loop (pink), DR (Y/H) motif (red), lid region (orange) and base sensing loop (yellow) are shown as thickened cartoon. (C) Cartoon of NDUFA10 with the density map (grey) from M. musculus CI bound to the Q-site inhibitor piericidin (PDB: 7B93). Mg²⁺ ATP and key binding residues are shown as sticks and colored by element. (D) Cartoon of NDUFA10 colored by atomic displacement parameter (ADP) in CI (PDB: 6ZKO). Red: most flexible, Blue: least flexible. (E,F) State dependent interactions between NDUFA5 and NDUFA10 involve the base-sensing loop. (E) Transparent surface of closed A-state CI showing NDUFA10 and NDUFA5 (auburn) as cartoons (PDB: 6ZKC). (F) Transparent surface of open D-state CI showing NDUFA10 and NDUFA5 as cartoons (PDB: 6ZKD). Key residues involved in the interaction are shown as sticks and colored by element. NDUFA10: cadet blue, NDUFA5: auburn.

FIGURE 10

TM accessory subunits of the ND2 and ND4 interface. The structure of mammalian CI (PDB: 6ZKC throughout) in cartoon and the surface of (A) NDUFC2 (green), (B) NDUFC1 (deep orange) and (C) NDUFB1 (red) shown, the core subunits colored as in Figure 1A and the accessory subunits grey. (D) Lipid binding pocket in the mammalian ND2 (thistle purple)/ND4 (pearly purple) interface with the accessory subunits in cartoon and lipids in spheres. (E) Lipid binding in the Y. lipolytica (PDB: 6YJ4) ND2/ND4 interface with the accessory subunits in cartoon and lipids in spheres. (F,G) NDUFC1 alters the structure of ND2 through lipid binding. (F) NDUFC2 and NDUFC1 form a cardiolipin binding pocket with the final TMH^ND2 in O. aries, TMH11^ND2 (PDB: 6ZKC). Positive residues that interact with the cardiolipin, as well as the cardiolipin, are represented as sticks. (G) The cardiolipin pocket is not present in Y. lipolytica which lacks NDUFC1 (PDB: 6YJ4). NDUFC2: green, NDUFC1: deep orange, ND2: thistle purple, ND4: pearly purple, NDUFB11: carrot orange, NDUFB5: cyan azure, NUXM: light cyan.

FIGURE 11

TM accessory subunits of the ND4 and ND5 interface. The structure of mammalian CI (PDB: 6ZKC throughout) in cartoon with the surface of (A) NDUFB11 (carrot orange), (B) NDUFB5 (cyan azure), and (C) NDUFB6 (old gold), the core subunits colored as in Figure 1A and the accessory subunits grey. (D) Lipid binding pocket in the mammalian ND4 (pearly purple)/ND5 (boysenberry purple) interface with the accessory subunits in cartoon and lipids in spheres. (E) Lipid binding in the Y. lipoyltica (PDB: 6YJ4) ND4/ND5 interface with the accessory subunits in cartoon and lipids in spheres. (F) The NDUFB11-S (short isoform) is shown in carrot orange cartoon. The additional 10 amino acids of the NDUFB11-L (long isoform, represented as dashed line) clash with core subunit ND4 but may be accommodated in the P_D-a′ assembly with accessory subunits NDUFB6, NDUFB10 (vivid auburn) and NDUFB11 shown in cartoon. NDUFB11: carrot orange, NDUFB5: cyan azure, NDUFB6: old gold, ND4: pearly purple, ND5: boysenberry purple, NDUFB10: vivid auburn.

FIGURE 12

The CX₉C motif containing accessory subunits. (A) The structure of mammalian CI (PDB: 6ZKC throughout unless stated otherwise) in cartoon with the surface of NDUFA8 (lilac purple), NDUFS5 (cadet blue), NDUFB10 (vivid auburn) and NDUFB7 (gold) shown, the core subunits colored as in Figure 1A and the other accessory subunits grey. (B) IMS view of the CI sole showing the CX₉C motif containing subunits. CI is shown as a transparent surface. The CX₉C subunits are shown as cartoons colored as in Figure 1 with the disulfide bonding residues shown as spheres. (C) Schematic diagram showing the import of CX₉C motif subunits into the IMS and assembly onto CI. CX₉C motif subunits are imported from the cytosol into the IMS via outer membrane protein TOM40. CX₉C subunits are oxidized by CHCHD4 protein bound to the AIF protein in its NADH bound state. The folded CX₉C subunits are assembled on their destined ETC complex. The electrons are transferred from CHCHD4 protein to CIV via the ALR protein and cyt c or are transferred from ALR protein to molecular O₂. (D) NDUFB10 interacts with the surface of ND4 (pearly purple) and compensates for the loss of the extended helical region of TMH14^ND4 present in bacteria. ND4 (NuoM) of E. coli CI (PDB: 7NZ1) and ND4 (Nqo13) of T. thermophilus (PDB: 4HEA) are superposed onto ND4 of mammalian (O. aries) CI structure. NDUFB10 and TMH14^ND4 are shown as cartoons. (E) NDUFB7 is myristoylated. Mammalian NDUFB7 (PDB: 6ZKO) of CI is shown as cartoon and other subunits are shown as colored surface. The myristoyl group is shown as spheres. NDUFA8: lilac purple, NDUFS5: cadet blue, NDUFB10: vivid auburn, NDUFB7: gold, ND4: pearly purple, ND5: boysenberry purple.

FIGURE 13

TM accessory subunits at the CI toe. The structure of mammalian CI (PDB: 6ZKC) in cartoon with the surface of (A) NDUFB2 (yellow orange) and (B) NDUFB3 (iguana green) shown, the core subunits colored as in Figure 1A and the other accessory subunits grey. (C) Cartoon of accessory subunits on surface of ND5 (boysenberry purple). Key residue mentioned in the text are shown. Lipid and NDUFB7 myristoyl group are shown as spheres. NDUFB2: yellow orange, NDUFB3: iguana green, ND5: boysenberry purple.

FIGURE 14

TM accessory subunits of the ND5 lateral helix (ND5 HL). The structure of mammalian CI (PDB: 6ZKC throughout unless stated otherwise) in cartoon with the surface of (A) NDUFB8 (vivid tangelo), (B) NDUFB4 (old gold) and (C) NDUFA11 (wheat) shown, the core subunits colored as in Figure 1A and the other accessory subunits grey. (D) Mammalian NDUFB8, NDUFB4 and NDUFA11 subunits are shown as cartoons on the surface of CI. Residues that are discussed in the text are shown as sticks. ND5 HL, TMH16^ND5 and TMH4^ND6 are shown as cartoon. Lipids are shown as spheres. (E) Zoom in on mammalian NDUFA11 shown as cartoon. ND5 HL, TMH16^ND5 and TMH4^ND6 are shown as cartoon. Lipids are shown as spheres. (F) Y. lipolytica NDUFA11 (NUJM; PDB: 6RFR) shown as cartoon. The ND5 (NU5M) HL (ND5 HL), TMH16^ND5(NU5M), TMH17^ND5(NU5M and TMH14^ND6(NU6M) are shown as cartoons. The lipids are shows as spheres. Subunits are colored as in Figure 1 unless stated otherwise. NDUFB8: vivid tangelo, NDUFB4: old gold, NDUFA11: wheat, ND5: boysenberry purple, ND6: cyan-blue.