Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states

Abstract

Complex I (NADH:ubiquinone oxidoreductase) uses the reducing potential of NADH to drive protons across the energy-transducing inner membrane and power oxidative phosphorylation in mammalian mitochondria. Recent cryo-EM analyses have produced near-complete models of all 45 subunits in the bovine, ovine and porcine complexes and have identified two states relevant to complex I in ischemia-reperfusion injury. Here, we describe the 3.3-Å structure of complex I from mouse heart mitochondria, a biomedically relevant model system, in the 'active' state. We reveal a nucleotide bound in subunit NDUFA10, a nucleoside kinase homolog, and define mechanistically critical elements in the mammalian enzyme. By comparisons with a 3.9-Å structure of the 'deactive' state and with known bacterial structures, we identify differences in helical geometry in the membrane domain that occur upon activation or that alter the positions of catalytically important charged residues. Our results demonstrate the capability of cryo-EM analyses to challenge and develop mechanistic models for mammalian complex I.

Figure 1. Overview of the structure of mouse complex I.

The fourteen core subunits are in color and labelled accordingly and the 26 supernumerary subunits are in white. The positions of the flavin mononucleotide (FMN), at the NADH binding site, and FeS cluster N2, the immediate electron donor to ubiquinone, are indicated. The structure of the cofactor chain, together with the modeled structure of ubiquinone-10, is shown separately in the inset also.

Figure 2. The resolution of densities for the core and supernumerary subunits in the 3.3 Å resolution map of active mouse complex I.

Local resolutions were estimated using the Local Resolution function in RELION with default parameters. The figure was created using UCSF Chimera, using the model to separate the core (a) and supernumerary (b) subunit densities.

Figure 3. Differences in structure between the active and deactive states of mouse complex I.

a) Left - NDUFA5, on the hydrophilic domain, and NDUFA10, on the membrane domain, form a second contact between the two domains. Right – the different arrangements of NDUFA5 and NDUFA10 are illustrated by superimposing the NDUFA10 subunit in the NDUFA5-NDUFA10 pairs from the bovine and mouse deactive and active states. NDUFA5 is present in markedly different positions in the active (red) and deactive (wheat and grey) states. b) Structural elements that become disordered or change between the active and deactive states. The ubiquinone binding channel determined by CAVER 3.0 is shown for reference. c) The different structures of ND6-TMH3 in the active (red) and deactive (wheat) states, superimposed on the N-terminal section, and the positions of key sidechains. The π-bulge is present only in the deactive state. d) Densities for key sidechains in the active state. e) Rotation of the bulky sidechains of Phe67 and Tyr69 between the TMHs of ND3, ND4L and ND1. The structures were superimposed on ND4L with the active structure colored by subunit and the deactive in wheat. Only the TMHs are shown, plus the loops between ND3-TMHs 1 and 2 and ND6-TMHs 3 and 4.

Figure 4. Phospholipids in the 3.3 Å resolution structure of mouse complex I.

a) Membrane-bound helices in the complex I viewed from the matrix side, with phospholipids in red and labelled with the nomenclature in Supplementary Table 3. b) Phospholipids stabilizing amphipathic helices on the matrix side of the heel of the complex. The structure shown in blue is from supernumerary subunits NDUFA1, NDUFA3, NDUFA7, NDUFA12 and NDUFA13 and phosphorus atoms are marked with spheres. c) Two phospholipids that interact with NDUFA9 may stabilize loops in ND3 and NDUFA9 that become disordered in the deactive state (shown in darker colors than the rest of the two subunits). The arrows on the insets to b) and c) indicate the directions of view.

Figure 5. Nucleotide/nucleoside binding to NDUFA10.

a) Density for the bound nucleotide/nucleoside (modeled as ADP) in NDUFA10 in active mouse complex I. The density has been carved around the modeled ADP and the phosphate in the overlaid model from panel c (2 Å carve radius, threshold level of 0.075). Key residues that interact with the nucleotide/nucleoside are indicated, and Asn36, the homolog of Ser36 is in green. b) Overview of the typical nucleoside-kinase fold of NDUFA10 with nucleotide bound and the P-loop adjacent to the bound phosphates. c) The nucleotide/nucleoside-binding site in bovine complex (taken from PDB 5O3121) with a phosphate added to the side-chain of Ser36 (green) on the basis of mass spectrometry evidence. The density has been carved around the phosphate and the ADP in the overlaid model from panel a (2 Å carve radius, threshold level of 0.16). d) Comparison of the binding mode of the ADP modeled in NDUFA10 (red) with deoxythymidine triphosphate bound in the feedback inhibition mode in Drosophila melanogaster deoxyribonucleoside kinase (PDB 1OE0, blue) and with ADP (green) and a nucleoside analog (wheat) in the substrate-binding site of human deoxycytidine kinase (PDB 2ZI435). The structures were overlaid using the whole nucleoside kinase fold structures.

Figure 6. The environment of cluster N2, the ubiquinone-binding channel and the E-channel in active mouse complex I.

a) Cluster N2 and nearby key residues in NDUFS2. Dimethyl-Arg85 and Arg105 are positively charged, His190 is the redox-Bohr group that changes protonation state upon N2 reduction,, and Asp104 and Glu115 may transfer protons to/from His190. b) The structure around the ubiquinone-headgroup binding site with key residues in NDUFS2. The structure of active mouse complex I (wheat) is overlaid on the modeled structure of bovine complex I with ubiquinone-10 bound (teal) in which hydrogen bonds are present between the headgroup and Tyr108 and His59, and His59 and Asp160. Distances in brackets are for the active mouse structure. c) The ubiquinone-binding channel (mesh) in active mouse complex I was predicted using CAVER 3.0 with a 1.4 Å probe radius. The key residues from panel b are shown with charged residues in the vicinity of the site (inset, charged residues around the center of the channel and their interactions). The main figure highlights the carboxylate-rich ND1 TMH 5-6 loop and the E channel residues (blue) that connect the ubiquinone-binding site to the π-helix present in ND6 in the deactive enzyme, and to charged residues in the central membrane plane. Long distances of interest are in red, and all residues are in ND1 unless otherwise indicated (B - NDUFS7, D - NDUFS2, J - ND6 and K - ND4L). All distances are in Å.

Figure 7. Charged residues and discontinuous and π-helices in subunits ND2, ND4 and ND5.

a) Overview of subunits ND2, ND4 and ND5 showing the layered arrangement of core TMHs 4 to 13. TMHs 4 to 6 (red) and 9 to 11 (magenta) are α helical whereas TMH7 is discontinuous, TMH8 contains π-helical structure (cyan) and TMH12 (blue) is also discontinuous. The transverse helix from ND5 is also shown. b) TMH8 in ND2, ND4 and ND5 orientated with the mitochondrial matrix at the top. c) Overlay of conserved charged residues in the central membrane plane in ND2, ND4 and ND5. A cartoon representation of ND4 is shown for reference, and conserved residues, are colored cyan for ND2, green for ND4 and pink for ND5. Numbers refer to ND4, except for residues only in ND5 numbered in grey. d) Comparison of structures of the transverse helix in ND5 with TMH16 (the C-terminus) on the right and the conserved aspartate residue indicated. Groups of structures have been aligned using the whole subunit structures. The ribbons join the Cα atoms together, and each fifth residue is numbered. The approximate positions of stretches of π-helix are indicated. e) The distribution of charged residues and helical elements along the membrane arm with key residues labelled and the distances between ionizable groups shown in Å. TMH7 and 12 are in red and TMH8 is in blue. Residues labeled in red are on TMH5, TMH7 and TMH12. The mouse structure is for the active state, the structure of T. thermophilus complex I is from PDB 4HEA and the structure of the membrane domain of complex I from E. coli is from PDB 3RKO.