Satoru Shimada

GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM

We developed a method, named GraDeR, which substantially improves the preparation of membrane protein complexes for structure determination by single-particle cryo-electron microscopy (cryo-EM). In GraDeR, glycerol gradient centrifugation is used for the mild removal of free detergent monomers and micelles from lauryl maltose-neopentyl glycol detergent stabilized membrane complexes, resulting in monodisperse and stable complexes to which standard processes for water-soluble complexes can be applied. We demonstrate the applicability of the method on three different membrane complexes, including the mammalian FoF1 ATP synthase. For this highly dynamic and fragile rotary motor, we show that GraDeR allows visualizing the asymmetry of the F1 domain, which matches the ground state structure of the isolated domain. Therefore, the present cryo-EM structure of FoF1 ATP synthase provides direct structural evidence for Boyers binding change mechanism in the context of the intact enzyme.

Complex structure of cytochrome c-cytochrome c oxidase reveals a novel protein-protein interaction mode

Mitochondrial cytochrome c oxidase (CcO) transfers electrons from cytochrome c (Cyt.c) to O2 to generate H2O, a process coupled to proton pumping. To elucidate the mechanism of electron transfer, we determined the structure of the mammalian Cyt.c–CcO complex at 2.0‐Å resolution and identified an electron transfer pathway from Cyt.c to CcO. The specific interaction between Cyt.c and CcO is stabilized by a few electrostatic interactions between side chains within a small contact surface area. Between the two proteins are three water layers with a long inter‐molecular span, one of which lies between the other two layers without significant direct interaction with either protein. Cyt.c undergoes large structural fluctuations, using the interacting regions with CcO as a fulcrum. These features of the protein–protein interaction at the docking interface represent the first known example of a new class of protein–protein interaction, which we term “soft and specific”. This interaction is likely to contribute to the rapid association/dissociation of the Cyt.c–CcO complex, which facilitates the sequential supply of four electrons for the O2 reduction reaction.

Purification of Active Respiratory Supercomplex from Bovine Heart Mitochondria Enables Functional Studies

To understand the roles of mitochondrial respiratory chain supercomplexes, methods for consistently separating and preparing supercomplexes must be established. To this end, we solubilized supercomplexes from bovine heart mitochondria with digitonin and then replaced digitonin with amphipol (A8–35), an amphiphilic polymer. Afterward, supercomplexes were separated from other complexes by sucrose density gradient centrifugation. Twenty-six grams of bovine myocardium yielded 3.2 mg of amphipol-stabilized supercomplex. The purified supercomplexes were analyzed based on their absorption spectra as well as Q10 (ubiquinone with ten isoprene units) and lipid assays. The supercomplex sample did not contain cytochrome c but did contain complexes I, III, and IV at a ratio of 1:2:1, 6 molecules of Q10, and 623 atoms of phosphorus. When cytochrome c was added, the supercomplex exhibited KCN-sensitive NADH oxidation; thus, the purified supercomplex was active. Reduced complex IV absorbs at 444 nm, so we measured the resonance Raman spectrum of the reduced amphipol-solubilized supercomplex and the mixture of amphipol-solubilized complexes I1, III2, and IV1 using an excitation wavelength of 441.6 nm, allowing measurement precision comparable with that obtained for complex IV alone. Use of the purified active sample provides insights into the effects of supercomplex formation.

Three-dimensional structure of bovine heart NADH: ubiquinone oxidoreductase (complex I) by electron microscopy of a single negatively stained two-dimensional crystal

Bovine heart NADH:ubiquinone oxidoreductase (complex I), which is the largest (about 1 MDa) membrane protein complex in the mitochondrial respiratory chain, catalyzes the electron transfer from NADH to ubiquinone, coupled with proton pumping. We have crystallized bovine complex I in reconstituted lipid bilayers and obtained a three-dimensional density map by the electron crystallographic analysis of a single negatively stained two-dimensional crystal. The asymmetric unit with dimensions of a = 388 Å, b = 129 Å and γ = 90° contains two molecules and is of P1 symmetry. Structural differences between the two molecules indicate flexibility of the hydrophilic domain relative to the membrane-embedded domain.

Structure of bovine cytochrome c oxidase in the ligand-free reduced state at neutral pH.

Although the enzymatic activity of cytochrome c oxidase (CcO) depends sensitively on pH over a wide range, X-ray structural analyses of bovine CcO have been conducted using crystals prepared at pH 5.7 owing to the difficulty in crystallizing this protein. Here, the structure of ligand-free reduced CcO was successfully determined at 1.99 Å resolution.

Structure of bovine cytochrome c oxidase crystallized at a neutral pH using a fluorinated detergent

Although the enzymatic activity of cytochrome c oxidase (CcO) depends sensitively on pH over a wide range, X-ray structure analyses of bovine CcO have been conducted using crystals prepared at pH 5.7 owing to difficulty in crystallizing this protein. Here, oxidized CcO at pH 7.3 was successfully crystallized using a fluorinated octyl-maltoside derivative, and the structure was determined at 1.77 Å resolution.

Solubilization conditions for bovine heart mitochondrial membranes allow selective purification of large quantities of respiratory complexes I, III, and V

Ascertaining the structure and functions of mitochondrial respiratory chain complexes is essential to understanding the biological mechanisms of energy conversion; therefore, numerous studies have examined these complexes. A fundamental part of that research involves devising a method for purifying samples with good reproducibility; the samples obtained need to be stable and their constituents need to retain the same structure and functions they possess when in mitochondrial membranes. Submitochondrial bovine heart particles were isolated using differential centrifugation to adjust to a membrane concentration of 46.0% (w/v) or 31.5% (w/v) based on weight. After 0.7% (w/v) deoxycholic acid, 0.4% (w/v) decyl maltoside, and 7.2% (w/v) potassium chloride were added to the mitochondrial membranes, those membranes were solubilized. At a membrane concentration of 46%, complex V was selectively solubilized, whereas at a concentration of 31.5% (w/v), complexes I and III were solubilized. Two steps-sucrose density gradient centrifugation and anion-exchange chromatography on a POROS HQ 20 μm column-enabled selective purification of samples that retained their structure and functions. These two steps enabled complexes I, III, and V to be purified in two days with a high yield. Complexes I, III, and V were stabilized with n-decyl-β-D-maltoside. A total of 200 mg-300 mg of those complexes from one bovine heart (1.1 kg muscle) was purified with good reproducibility, and the complexes retained the same functions they possessed while in mitochondrial membranes.

A unique respiratory adaptation in Drosophila independent of supercomplex formation

Large assemblies of respiratory chain complexes, known as supercomplexes, are present in the mitochondrial membrane in mammals and yeast, as well as in some bacterial membranes. The formation of supercomplexes is thought to contribute to efficient electron transfer, stabilization of each enzyme complex, and inhibition of reactive oxygen species (ROS) generation. In this study, mitochondria from various organisms were solubilized with digitonin, and then the solubilized complexes were separated by blue native PAGE (BN-PAGE). The results revealed a supercomplex consisting of complexes I, III, and IV in mitochondria from bovine and porcine heart, and a supercomplex consisting primarily of complexes I and III in mitochondria from mouse heart and liver. However, supercomplexes were barely detectable in Drosophila flight-muscle mitochondria, and only dimeric complex V was present. Drosophila mitochondria exhibited the highest rates of oxygen consumption and NADH oxidation, and the concentrations of the electron carriers, cytochrome c and quinone were higher than in other species. Respiratory chain complexes were tightly packed in the mitochondrial membrane containing abundant phosphatidylethanolamine with the fatty acid palmitoleic acid (C16:1), which is relatively high oxidation-resistant as compared to poly-unsaturated fatty acid. These properties presumably allow efficient electron transfer in Drosophila. These findings reveal the existence of a new mechanism of biological adaptation independent of supercomplex formation.

Structure and function of the intermembrane space domain of mammalian FoF1 ATP synthase

Mitochondrial FoF1 ATP synthase is a membrane bound molecular machine central to cellular energy conversion and cristae architecture. Recently, a novel domain has been visualized in the intermembrane space region of mammalian ATP synthase. The complete three-dimensional (3D) structure, composition and function of this domain - which we term intermembrane space domain (IMD) - are unknown. Here, we present two distinct 3D structures of monomeric bovine FoF1 ATP synthase by single particle cryo-electron microscopy (cryo-EM) that differ by the presence and absence of the IMD. Comparison of both structures reveals the IMD to be a bipartite and weakly associated domain of FoF1 ATP synthase. The tubular sub-domain of the IMD appears to contact the rotor-ring region, its globular sub-domain is anchored in the membrane-bending kink of the ATP synthase. However, absence of the IMD does not impact the kink in the transmembrane region ruling out a functional role in membrane bending. By combining our structural analysis with chemical cross-linking and reported biochemical, genetic and structural data we identify 6.8PL and DAPIT as the subunits forming the intermembrane space domain. We compare the present structure of the mammalian IMD in the bovine FoF1 ATP synthase monomer with structures of dimeric FoF1 ATP synthase from yeast and ciliate showing that the IMD is a common, but structurally divergent feature of several mitochondrial ATP synthases. On the basis of our analysis we discuss potential functions of the novel domain in rotary catalysis, oligomerization and mitochondrial permeability transition.