Inhibition of ATP synthase reverse activity restores energy homeostasis in mitochondrial pathologies.

The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.

Large-scale column-free purification of bovine F-ATP synthase.

Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers, tetramers, dimers, and monomers. In vitro investigations of mammalian F-ATP synthase are often limited by the ability to purify the oligomeric forms present in vivo at a quantity, stability, and purity that meets the demand of the planned experiment. We developed a purification approach for the isolation of bovine F-ATP synthase from heart muscle mitochondria that uses a combination of buffer conditions favoring inhibitor factor 1 binding and sucrose density gradient ultracentrifugation to yield stable complexes at high purity in the milligram range. By tuning the glyco-diosgenin to lauryl maltose neopentyl glycol ratio in a final gradient, fractions that are either enriched in tetrameric or monomeric F-ATP synthase can be obtained. It is expected that this large-scale column-free purification strategy broadens the spectrum of in vitro investigation on mammalian F-ATP synthase.

NDT-C11 as a viable novel detergent for single particle cryo-EM.

Single particle cryo electron microscopy (cryo-EM) is now the major method for the determination of integral membrane protein structure. For the success of a given project the type of membrane mimetic used for extraction from the native cell membrane, purification to homogeneity and finally cryo-grid vitrification is crucial. Although small molecule amphiphiles - detergents - are the most widely used membrane mimetic, specific tailoring of detergent structure for single particle cryo-EM is rare and the demand for effective detergents not satisfied. Here, we compare the popular detergent lauryl maltose-neopentyl glycol (LMNG) with the novel detergent neopentyl glycol-derived triglucoside-C11 (NDT-C11) in its behavior as free detergent and when bound to two types of multisubunit membrane protein complexes - cyanobacterial photosystem I (PSI) and mammalian F-ATP synthase. We conclude that NDT-C11 has high potential to become a very useful detergent for single particle cryo-EM of integral membrane proteins.

Two-dimensional crystallization of intact F-ATP synthase isolated from bovine heart mitochondria.

Mitochondrial F-ATP synthase produces the majority of ATP for cellular functions requiring free energy. The structural basis for proton motive force-driven rotational catalysis of ATP formation in the holoenzyme remains to be determined. Here, the purification and two-dimensional crystallization of bovine heart mitochondrial F-ATP synthase are reported. Two-dimensional crystals of up to 1 µm in size were grown by dialysis-mediated detergent removal from a mixture of decylmaltoside-solubilized 1,2-dimyristoyl-sn-glycero-3-phosphocholine and F-ATP synthase against a detergent-free buffer. A projection map calculated from an electron micrograph of a negatively stained two-dimensional crystal revealed unit-cell parameters of a = 185.0, b = 170.3 Å, γ = 92.5°.

Rapid and Highly Stable Membrane Reconstitution by LAiR Enables the Study of Physiological Integral Membrane Protein Functions.

Functional reintegration into lipid environments represents a major challenge for in vitro investigation of integral membrane proteins (IMPs). Here, we report a new approach, termed LMNG Auto-insertion Reintegration (LAiR), for reintegration of IMPs into lipid bilayers within minutes. The resulting proteoliposomes displayed an unprecedented capability to maintain proton gradients and long-term stability. LAiR allowed for monitoring catalysis of a membrane-bound, physiologically relevant polyisoprenoid quinone substrate by Escherichia coli cytochromes bo 3 (cbo 3) and bd (cbd) under control of the proton motive force. LAiR also facilitated bulk-phase detection and physiological assessment of the "proton leak" in cbo 3, a controversial catalytic state that previously was only approachable at the single-molecule level. LAiR maintained the multisubunit integrity and higher-order oligomeric states of the delicate mammalian F-ATP synthase. Given that LAiR can be applied to both liposomes and planar membrane bilayers and is compatible with IMPs and lipids from prokaryotic and eukaryotic sources, we anticipate LAiR to be applied broadly across basic research, pharmaceutical applications, and biotechnology.

Purified F-ATP synthase forms a Ca2+-dependent high-conductance channel matching the mitochondrial permeability transition pore.

The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca2+ dissipates the H+ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca2+ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg2+ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca2+ can transform the energy-conserving F-ATP synthase into an energy-dissipating device.

Structural Basis for Selective Binding of Export Cargoes by Exportin-5.

In the nucleus, RanGTP binding to importin dissociates the cargo. On the other hand, RanGTP enables exportin to bind export cargo and form the export complex by each exportin's own cargo selection mechanism. Here, we present two X-ray structures for Exportin-5 (Exp-5) alone and Exp-5:RanGTP intermediate complex. The structure of Exp-5 adopts a ring-shaped closed conformation by C-terminal anchor residues 1,167-1,179, interacting with N-terminal heat repeats 4-9. The closed form of Exp-5 is important for the stability of the cargo-free state. Interaction between Exp-5 and RanGTP induces elimination of intramolecular contacts of the C-terminal anchor. A large movement of N-terminal 1-9th heat repeats and C-terminal 19-20th heat repeats creates an open space for RanGTP accommodation. Exp-5 in Exp-5:RanGTP and Exp-5:RanGTP:pre-miRNA adopts the same conformation. RanGTP binding to Exp-5 creates a selective molecular cage area for accepting its cargoes, such as small double-stranded RNAs, without conformational change in Exp-5:RanGTP.

Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry.

Membrane proteins reside in lipid bilayers and are typically extracted from this environment for study, which often compromises their integrity. In this work, we ejected intact assemblies from membranes, without chemical disruption, and used mass spectrometry to define their composition. From Escherichia coli outer membranes, we identified a chaperone-porin association and lipid interactions in the ß-barrel assembly machinery. We observed efflux pumps bridging inner and outer membranes, and from inner membranes we identified a pentameric pore of TonB, as well as the protein-conducting channel SecYEG in association with F1FO adenosine triphosphate (ATP) synthase. Intact mitochondrial membranes from Bos taurus yielded respiratory complexes and fatty acid-bound dimers of the ADP (adenosine diphosphate)/ATP translocase (ANT-1). These results highlight the importance of native membrane environments for retaining small-molecule binding, subunit interactions, and associated chaperones of the membrane proteome.

Bovine F1Fo ATP synthase monomers bend the lipid bilayer in 2D membrane crystals.

We have used a combination of electron cryo-tomography, subtomogram averaging, and electron crystallographic image processing to analyse the structure of intact bovine F(1)F(o) ATP synthase in 2D membrane crystals. ATPase assays and mass spectrometry analysis of the 2D crystals confirmed that the enzyme complex was complete and active. The structure of the matrix-exposed region was determined at 24 Å resolution by subtomogram averaging and repositioned into the tomographic volume to reveal the crystal packing. F(1)F(o) ATP synthase complexes are inclined by 16° relative to the crystal plane, resulting in a zigzag topology of the membrane and indicating that monomeric bovine heart F(1)F(o) ATP synthase by itself is sufficient to deform lipid bilayers. This local membrane curvature is likely to be instrumental in the formation of ATP synthase dimers and dimer rows, and thus for the shaping of mitochondrial cristae.

Selective nuclear export mechanism of small RNAs.

The timely nuclear-cytoplasmic translocation of proteins and RNAs by importins and exportins is important for controlling biological processes. Since the 2004 publication of the first exportin structure, Cse1p, the X-ray structures of exportin-5 complexed with pre-microRNA, exportin-t complexed with tRNA, and three CRM1-related structures have revealed the binding mechanism involved in specific cargo recognition. Pre-microRNA and tRNA have conserved 3' 2-4-nucleotide overhang motifs and similar short double-stranded regions. Exportin-5 and exportin-t bind a conserved 3' overhang strongly, and they weakly enclose the short double-stranded stems, each in a different manner. The structures of the nuclear export complexes of small double-stranded RNAs, pre-microRNAs, and tRNAs provide information about the specificities of the two exportins in the context of transcription and translation control.