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Structure of mammalian respiratory complex I

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 subunits1,2. 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 hypoxia3,4. Our structures therefore provide a foundation for understanding complex I assembly5 and the effects of mutations that cause clinically relevant complex I dysfunctions6, give insights into the structural and functional roles of the supernumerary subunits and reveal new information on the mechanism and regulation of catalysis.

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Figure 1: The supernumerary subunits of mammalian complex I.
Figure 2: Details of some of the supernumerary subunits.
Figure 3: The core subunits and ubiquinone-binding site of mammalian complex I.
Figure 4: Relationships between classes 1 and 2.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The electron microscopy maps and models for each class have been deposited in the Electron Microscopy Data Bank (EMDB) with accession numbers EMD-4040 (class 1), EMD-4032 (class 2) and EMD-4041 (class 3), and in the Protein Data Bank with accessions 5LDW (class 1), 5LC5 (class 2) and 5LDX (class 3).

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Acknowledgements

We thank R. Henderson and G. Murshudov for discussions, J. Grimmett and T. Darling for computational help, S. Chen and C. Savva for electron microscopy help, and J. N. Blaza for quantifying the ratio of de-active and active enzymes in our preparation. This work was supported by The Medical Research Council, grant numbers U105184322 (K.R.V., in R. Henderson’s group) and U105663141 (J.H.).

Author information

Authors and Affiliations

Authors

Contributions

J.Z. prepared protein; K.R.V. carried out electron microscopy data collection and analysis with help from J.Z.; J.Z. built the initial model; J.Z., K.R.V. and J.H. worked together, led by J.H., to model and analyse the data; J.H. designed the project; J.H. wrote the paper with help from J.Z. and K.R.V.

Corresponding authors

Correspondence to Kutti R. Vinothkumar or Judy Hirst.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information

Nature thanks R. B. Gennis, M. T. Ryan and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data 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 ResMap35. 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 in the 7−9 Å resolution range dominates the lower-resolution features. 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.

Extended Data Figure 2 Resolution estimation and ResMap analysis of the classes of complex I.

ac, 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.

Extended Data 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.

Extended Data 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, The 15 kDa subunit on the IMS face, containing a CHCH domain with two disulfide 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 modelled).

Extended Data 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 Extended Data 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 the 49 kDa subunit and the transverse helix in ND5, as well as elements of ND4, ND6, B14.7, ESSS and B15.

Extended Data Table 1 Summary of the models for the core subunits of B. taurus complex I
Extended Data Table 2 Summary of the models for the supernumerary subunits of B. taurus complex I
Extended Data Table 3 Subunit–subunit interactions for the supernumerary subunits
Extended Data Table 4 Allocation of subunits to domains, and the relative movement of domains between classes 1, 2 and 3
Extended Data Table 5 Data collection, refinement and model statistics for classes 1 and 2

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Zhu, J., Vinothkumar, K. & Hirst, J. Structure of mammalian respiratory complex I. Nature 536, 354–358 (2016). https://doi.org/10.1038/nature19095

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