Structural basis of respiratory complex adaptation to cold temperatures

Abstract

In response to cold, mammals activate brown fat for respiratory-dependent thermogenesis reliant on the electron transport chain. Yet, the structural basis of respiratory complex adaptation upon cold exposure remains elusive. Herein, we combined thermoregulatory physiology and cryoelectron microscopy (cryo-EM) to study endogenous respiratory supercomplexes from mice exposed to different temperatures. A cold-induced conformation of CI:III₂ (termed type 2) supercomplex was identified with a ∼25° rotation of CIII₂ around its inter-dimer axis, shortening inter-complex Q exchange space, and exhibiting catalytic states that favor electron transfer. Large-scale supercomplex simulations in mitochondrial membranes reveal how lipid-protein arrangements stabilize type 2 complexes to enhance catalytic activity. Together, our cryo-EM studies, multiscale simulations, and biochemical analyses unveil the thermoregulatory mechanisms and dynamics of increased respiratory capacity in brown fat at the structural and energetic level.

Keywords: CIII(2) rotation; brown adipose tissue; cellular adaptation; electron transport chain; membrane lipid remodeling; respiratory complexes.

Figure 1.. Identification of type 2 CI:CIII₂ complexes in cold-exposed mouse iBAT.

(A) Schematic workflow for mitochondrial membrane isolation (see STAR Methods). Mice were housed at room temperature and exposed to either thermoneutral or cold conditions for 8 days. Interscapular brown fat (iBAT) was isolated, and their mitochondria separated by ultracentrifugation. Mitochondria were sonicated and membranes collected, resuspended in digitonin buffer and complexes separated by size exclusion chromatography before EM analyses. (B) Blue-Native PAGE of isolated complexes. (C) Size exclusion chromatogram and representative negative stain EM images of each group. Red framed fractions contain respiratory complexes.

Figure 2.. Rotation of CIII₂ in type 2 complexes upon cold exposure.

(A) EM density maps (4σ contour) depict the rotation angle of CIII₂ along CI longitudinal MD axis. Type 1 (hot colors, left) and 2 (cold colors, right) complexes as seen from the mitochondrial matrix side. Each CIII₂ protomer is colored differently to appreciate contacts with CI. Angles were calculated as the intersection of CIII₂ transdimer axis with CI membrane domain central axis. (B-D), 3D rotation of CIII₂ in type 2 complexes compared to type 1^WT-TN from the matrix (B), side (C) and rear (D) views. Angles are calculated by comparing CIII₂ interdimer or cross-sectional axes in type 2 to those in CIII₂ in type 1. IMM; inner mitochondrial membrane. IMS; intermembrane space between OMM (outer mitochondrial membrane) and IMM. Matrix; matrix compartment of mitochondria. (E) Data from 3 x 1 μs molecular dynamics simulations. Top, kernel density estimation (KDE) of the angle between the two vectors from all MD simulation replicas of type 1 and type 2. The first vector passes through the membrane arm of CI and the second from the distal to the proximal UQCRC1 subunit of CIII₂. Bottom, kernel density estimation (KDE) of the angle between the z-axis vector with the vector going through the CIII₂. Green lines indicate the vectors for the calculated angles.

Figure 3.. Organization of type 2 CI:CIII₂ complexes.

(A) Lipophilicity (golden) across the side view of CI:III₂ classes shows the transmembrane domain belt that defines straightening of type 2 compared to angled type 1 complexes. (B) Rotation of CIII₂ in type 2 complexes approximates UQCRB (CIII₂) to NDUFA9 (CI) and forms a hydrophobic protein bridge at the CoQ-channel. (C) The kernel density estimation (KDE) of the distance between center of mass of UQCRB and NDUFA9 for all three simulation replicas of type 1 and type 2 complexes. Data shown is from 3 x 1μs molecular dynamics simulations of each type 1 and type 2. (D) Representation of CIII₂ CoQ exchange cavities in type 1 (hot colors) and type 2 (cold colors) complexes. Type 2 complexes show complete exposure of both cavities from the CI CoQ-site view (orange). In B and D, red arrow indicates the displacement distance relative to type 1 complexes.

Figure 4.. Protein-lipid dynamics in type 2 CI:III₂ complexes.

(A) Lipid composition in isolated mitochondria and detail of species with q<0.05 from WT (PERK^+/+) mice under thermoneutral (TN) or cold conditions and PERK KO (PERK^−/−) mice under cold. (B) Lipid composition of isolated mitochondrial-associated membranes WT (PERK^+/+) mice under thermoneutral (TN) or cold conditions and (PERK^−/−) mice under cold. Schematic representation of PC and PE species synthesis in ER and mitochondria. (C) GO enrichment from mitochondrial-ER proteomics with key enzymes in phospholipid synthesis. (D) Lipid occupancy (isosurface shown at threshold level 0.5 from ChimeraX) in type 1 and type 2 complexes during 3 x 1μs molecular dynamics simulations and depiction of the number of contacting lipids and stabilization of CIII₂. Top graph, number of lipids at the interface of CI and CIII₂ (based on the radial distance of 15 Å CI and CIII₂. Bottom graph, the kernel density estimation (KDE) of the RMSD of CIII₂, when the system is aligned based on the membrane arm of complex I for all simulation replicas of type 1 and type 2 complexes. (E) Representation of protein-lipid dynamics over 1000 ns. (F) Quantitation of lipid contacts within 5 – of the protein from the experiments in panel (E).

Figure 5.. Dynamics of CI and CIII₂ structures in type 2 assemblies.

(A) Michaelis-Menten kinetics of isolated CI:CIII2 complexes from iBAT exposed to different thermal conditions. TN; thermoneutral. N=3, n=3. (B) Apparent Michaelis-Menten parameters from the experiments represented in (A). (C) Depiction of maximum opening and closure of CI peripheral arm during 1 μs molecular dynamics simulations. Green lines indicate the vectors for the calculated angles. Also, shown is the kernel density estimation (KDE) of the angle between PA and MD of CI from 3 x 1μs simulation data of type 1 and 2 complexes. (D) Representation of the active site of CI across different open and closed states in type 2 complexes. (E) EM density maps of proximal UQCRFS1 (Rieske) head and neck (green circle) from type 2 complexes show decreased EM densities (red) as seen from the side and IMS views of CIII₂ (3σ contour). (F) Type 2 complexes display increased flexibility of the cytochrome C1 subunit acidic loop (150-170) close to proximal UQCRFS1 subunit and approximates to the same loop in the adjacent protomer. (G) Schematic representation of conformational dynamics in thermoneutral (TN) and cold conditions. An energy barrier in TN-like conditions is minimized in cold-like conditions. As a result of the loss of barrier (blue profile), the type 2 conformation is observed, otherwise not. The low energy type 2 arrangement enhances the conformational variation in cold compared to TN conditions.

Figure 6.. Structural mechanisms of enhanced CI:III₂ catalytic activity in cold-induced type 2 complexes.

In response to low environmental temperatures, brown adipocytes increase thermogenic function to maintain body temperature. This depends on increased mitochondrial energetics where respiratory CI:III₂ complexes rearrange from their canonical conformation (type 1) to a non-canonical (type 2) where CIII₂ is rotated. Type 2 complexes contain protein-lipid contacts which may be favored by cold-dependent remodeling of mitochondrial membrane lipid species. Type 2 complexes show enhanced catalytic efficiency through different mechanisms: 1) CI dehydrogenase activity appears augmented due to higher tilting in narrower angles (open/closed) which indicates higher turnover rates; 2) the distances between CI and CIII₂ are shortened, facilitating CoQ-channeling; and 3) CIII₂ increases the transfer of electrons from CI reduced coenzyme Q (CoQH2) to Cytochrome C (CYT C). As a result of increased CI:CIII₂ activities, a high proton gradient is generated at the IMS which is dissipated by the uncoupling protein UCP1 to generate heat that maintains body temperature. IMS; intermembrane space. IMM; inner mitochondrial membrane. Image created with BioRender.com.