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. 2016 Aug 18;536(7616):354-358.
doi: 10.1038/nature19095. Epub 2016 Aug 10.

Structure of Mammalian Respiratory Complex I

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Free PMC article

Structure of Mammalian Respiratory Complex I

Jiapeng Zhu et al. Nature. .
Free PMC article

Abstract

Complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in the cell, powers ATP synthesis in mammalian mitochondria by using the reducing potential of NADH to drive protons across the inner mitochondrial membrane. Mammalian complex I (ref. 1) contains 45 subunits, comprising 14 core subunits that house the catalytic machinery (and are conserved from bacteria to humans) and a mammalian-specific cohort of 31 supernumerary subunits. Knowledge of the structures and functions of the supernumerary subunits is fragmentary. Here we describe a 4.2-Å resolution single-particle electron cryomicroscopy structure of complex I from Bos taurus. We have located and modelled all 45 subunits, including the 31 supernumerary subunits, to provide the entire structure of the mammalian complex. Computational sorting of the particles identified different structural classes, related by subtle domain movements, which reveal conformationally dynamic regions and match biochemical descriptions of the 'active-to-de-active' enzyme transition that occurs during hypoxia. Our structures therefore provide a foundation for understanding complex I assembly and the effects of mutations that cause clinically relevant complex I dysfunctions, give insights into the structural and functional roles of the supernumerary subunits and reveal new information on the mechanism and regulation of catalysis.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

ED Figure 1
ED Figure 1. Resolution estimation and ResMap analysis of the density map for complex I before classification.
a) The map, shown at two different contour levels, is coloured according to the local resolution, as determined by ResMap. At the higher contour level (left), the majority of the protein is resolved to 3.9–4.7 Å; only the very peripheral regions (parts of the 51 kDa, 24 kDa and 75 kDa subunits in the matrix arm, and the distal end of the membrane arm) are at lower resolutions of 5–6 Å. At the lower contour level (right), the detergent/lipid belt dominates the 7–9 Å resolution range. b) A slice through the map shows that large portions of the central, core regions are resolved to 4 Å or better. c) The FSC curve defines an estimated overall resolution of 4.16 Å at FSC = 0.143.
ED Figure 2
ED Figure 2. Resolution estimation and ResMap analysis of the classes of complex I.
Panels a, b and c show data on classes 1, 2 and 3, respectively. Classes 1 and 2 display similar distributions in local resolution, with the majority of the protein in the range 4–5 Å. In class 3 the majority of protein displays a resolution of 4.5–5 Å. In all three cases the refined models agree very well with the maps as shown by comparison of the FSC curves (red) and the FSC curves from the half-maps (blue), and the similarity of the resolution values at FSC 0.143 and 0.5. The estimated resolutions, defined where the line at FSC = 0.143 crosses the blue curve, are 4.27 Å for class 1, 4.35 Å for class 2 and 5.6 Å for class 3. d). Cross-validation of the refinement parameters, confirming lack of over-fitting. For classes 1 and 2, one of the half maps was used for refinement then the FSC curves were calculated for each of the two half maps using the same model.
ED Figure 3
ED Figure 3. Example regions of the cryoEM density map for the core subunits, and the model fitted to the map.
a) A selection of TMHs from the membrane domain: TMH3 from ND1, the distorted TMHs in ND6, ND4 and ND5, and a discontinuous TMH from ND2. The series of TMHs from left to right illustrates the decrease in resolution along the domain. b) The two [4FeS4] clusters in the 75 kDa subunit (density in red, at higher contour level) with the protein ligating one of them. c) The FMN cofactor in the 51 kDa subunit. d) The β-sheet in subunit PSST, showing clear separation between the strands. e) Two helices from the 49 kDa subunit.
ED Figure 4
ED Figure 4. Example regions of the cryoEM density map for the supernumerary subunits, and the model fitted to the map.
a) Subunit MWFE, containing one TMH. b) Subunit B14.5, containing two TMHs. The N- and C-terminal loops are not shown. c) 15 kDa subunit on the IMS face, containing a CHCH domain with two disulphide bonds. The N- and C-terminal loops are not shown. d) The seven-strand β-sheet in the 39 kDa subunit, showing the separation of the strands, and the bound nucleotide (red density) modelled as NADPH. e) Helix 1, one of the arginine-rich helices, in B22, and SDAP-β, on the matrix side of the tip of the membrane domain. Inset: the weak density attached to Ser44 in SDAP-β attributed to the attached pantetheine-4'-phosphate group (side chain of Ser44 not shown).
ED Figure 5
ED Figure 5. Relationships between classes 1 and 3.
a) The structures for classes 1 and 3 have been superimposed using ND1 and ND3. In class 3, relative to class 1, the hydrophilic and distal membrane domains are both rotated and shifted, but the change in the membrane domain dominates. Although the changes appear to make the angle of the L-shaped molecule increase they do not originate from a hinge-like motion at the interface of the hydrophilic and membrane domains. Class 1 is in red, class 3 is in red (ND1 domain), wheat, blue and cyan. Details of the composition and movement of the domains are given in ED Table 4. b) The density for class 3 (white) is presented with the model for well-resolved regions of class 3 in blue (the model is enclosed in the density) and the model for poorly resolved regions in red (the model appears outside the density). The poorly resolved regions include the N-terminus of 49 kDa and the transverse helix in ND5, as well as elements of ND4, ND6, B14.7, ESSS and B15.
Figure 1
Figure 1. The supernumerary subunits of mammalian complex I.
a) Overview of the complex with the 14 core subunits in blue (FeS clusters in yellow/orange), the 31 supernumerary subunits in red, and the cryoEM density in grey. b-c) Arrangement and structures of the supernumerary subunits around the core membrane (b) and hydrophilic (c) subunits. The core subunits are in surface representation and the supernumerary subunits in cartoon; icons show viewpoints and locations in the complex. Subunits in brackets are behind the domain. The assignments of B12 and AGGG may be reversed. See ED Tables 1 and 2 for the subunit nomenclatures in other species.
Figure 2
Figure 2. Details of some of the supernumerary subunits.
a) Subunits confined to the IMS face with disulphide bonds indicated by red spheres. b) Positively-charged residues in the LYR-protein B14 (blue) interact with negatively-charged residues in SDAP-α (red); B22 and SDAP-β exhibit the same structural motif. c) 42 kDa with the core nucleoside-kinase fold in rainbow; the extensions (grey) dock it to ND2. d) 39 kDa with the core dehydrogenase-reductase fold in rainbow and bound nucleotide; the C-terminal domain (grey) approaches the membrane interface. Icons indicate viewpoints and locations in the complex.
Figure 3
Figure 3. The core subunits and ubiquinone-binding site of mammalian complex I.
a) The core subunits with the FMN, FeS clusters, conserved charged residues (Cα) in the membrane (overlaid for clarity), and proposed ubiquinone-binding channel (orange). b) The ubiquinone-binding channel. 49 kDa-Tyr108 and 49 kDa-His59 form hydrogen bonds to the bound ubiquinone at the top of the cleft (indicated by an arrow) between 49 kDa and PSST. The channel entrance is between three helices in ND1. c) Structural elements forming the channel and bottleneck (between the arrows). PSST-Arg77 is hydroxylated. d) Conformations of the 49 kDa (β1-β2) and ND1 (TMH5-6) loops observed in different species. In T. thermophilus the ubiquinone headgroup binds between Tyr108 and His59 and His59 hydrogen bonds to 49 kDa-Asp160. In Y. lipolytica a quinazoline inhibitor is bound between PSST-Met60 and the tip of the 49 kDa loop.
Figure 4
Figure 4. Relationships between classes 1 and 2.
a) Class 1 (red) and 2 (blue/wheat) were superimposed using ND1+ND3 and viewed along the axis of rotation for ND4+ND5. See ED Table 4 for details of the transformations. b) Change of ~10 Å in the relative positions of B14+SDAPα (hydrophilic domain) and 42 kDa (membrane domain); class 1 in red, viewed from the matrix. c) Loops (ND1 TMH5-6, ND3 TMH1-2, 49 kDa β1-β2 and in 39 kDa) in class 2 that are disordered in class 1, with the ubiquinone-binding site; adjacent TMHs/strands are shown for clarity. The site cannot be detected in class 1 as it appears open. d) Densities for the loop connecting ND3 TMH1-2. For class 1, the loop from the class 2 model (white) was used to identify the density in red.

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