Mechanism of proton-powered c-ring rotation in a mitochondrial ATP synthase.

Proton-powered c-ring rotation in mitochondrial ATP synthase is crucial to convert the transmembrane protonmotive force into torque to drive the synthesis of adenosine triphosphate (ATP). Capitalizing on recent cryo-EM structures, we aim at a structural and energetic understanding of how functional directional rotation is achieved. We performed multi-microsecond atomistic simulations to determine the free energy profiles along the c-ring rotation angle before and after the arrival of a new proton. Our results reveal that rotation proceeds by dynamic sliding of the ring over the a-subunit surface, during which interactions with conserved polar residues stabilize distinct intermediates. Ordered water chains line up for a Grotthuss-type proton transfer in one of these intermediates. After proton transfer, a high barrier prevents backward rotation and an overall drop in free energy favors forward rotation, ensuring the directionality of c-ring rotation required for the thermodynamically disfavored ATP synthesis. The essential arginine of the a-subunit stabilizes the rotated configuration through a salt bridge with the c-ring. Overall, we describe a complete mechanism for the rotation step of the ATP synthase rotor, thereby illuminating a process critical to all life at atomic resolution.

Insight into neofunctionalization of 2,3-oxidosqualene cyclases in B,C-ring-opened triterpene biosynthesis in quinoa.

Bioactive triterpenes feature complex fused-ring structures, primarily shaped by the first-committed enzyme, 2,3-oxidosqualene cyclases (OSCs) in plant triterpene biosynthesis. Triterpenes with B,C-ring-opened skeletons are extremely rare with unknown formation mechanisms, harbouring unchartered chemistry and biology. Here, through mining the genome of Chenopodium quinoa followed by functional characterization, we identified a stress-responsive and neofunctionalized OSC capable of generating B,C-ring-opened triterpenes, including camelliol A and B and the novel (-)-quinoxide A as wax components of the specialized epidermal bladder cells, namely the quinoxide synthase (CqQS). Protein structure analysis followed by site-directed mutagenesis identified key variable amino acid sites underlying functional interconversion between pentacyclic ß-amyrin synthase (CqbAS1) and B,C-ring-opened triterpene synthase CqQS. Mutation of one key residue (N612K) in even evolutionarily distant Arabidopsis ß-amyrin synthase could generate quinoxides, indicating a conserved mechanism for B,C-ring-opened triterpene formation in plants. Quantum computation combined with docking experiments further suggests that conformations of conserved W613 and F413 of CqQS might be key to selectively stabilizing intermediate carbocations towards B,C-ring-opened triterpene formation. Our findings shed light on quinoa triterpene skeletal diversity and mechanisms underlying B,C-ring-opened triterpene biosynthesis, opening avenues towards accessing their chemistry and biology and paving the way for quinoa trait engineering and quality improvement.

Two new cassane diterpenoids from the seed kernels of Caesalpinia sinensis.

Two new cassane diterpenoids, sucupiranin MN (1) and sucupiranin ML (2), together with two known compounds sucutinirane C (3) and deacetylsucutinirane C (4) were isolated from the seed kernels of Caesalpinia sinensis. Their structures were elucidated by means of analysis of comprehensive spectroscopic data, especially HRESIMS and 1D/2D NMR spectroscopy. Compounds 1-4 are typical furan-type cassane derivatives with an aromatized C ring. Biological evaluation revealed that compounds 1-4 at the concentration of 10 µM could inhibit the overproduction of NO in LPS-stimulated RAW 264.7 macrophages.

ATP yield of plant respiration: potential, actual and unknown.

BACKGROUND AND AIMS: The ATP yield of plant respiration (ATP/hexose unit respired) quantitatively links active heterotrophic processes with substrate consumption. Despite its importance, plant respiratory ATP yield is uncertain. The aim here was to integrate current knowledge of cellular mechanisms with inferences required to fill knowledge gaps to generate a contemporary estimate of respiratory ATP yield and identify important unknowns. METHOD: A numerical balance sheet model combining respiratory carbon metabolism and electron transport pathways with uses of the resulting transmembrane electrochemical proton gradient was created and parameterized for healthy, non-photosynthesizing plant cells catabolizing sucrose or starch to produce cytosolic ATP. KEY RESULTS: Mechanistically, the number of c subunits in the mitochondrial ATP synthase Fo sector c-ring, which is unquantified in plants, affects ATP yield. A value of 10 was (justifiably) used in the model, in which case respiration of sucrose potentially yields about 27.5 ATP/hexose (0.5 ATP/hexose more from starch). Actual ATP yield often will be smaller than its potential due to bypasses of energy-conserving reactions in the respiratory chain, even in unstressed plants. Notably, all else being optimal, if 25 % of respiratory O2 uptake is via the alternative oxidase - a typically observed fraction - ATP yield falls 15 % below its potential. CONCLUSIONS: Plant respiratory ATP yield is smaller than often assumed (certainly less than older textbook values of 36-38 ATP/hexose) leading to underestimation of active-process substrate requirements. This hinders understanding of ecological/evolutionary trade-offs between competing active processes and assessments of crop growth gains possible through bioengineering of processes that consume ATP. Determining the plant mitochondrial ATP synthase c-ring size, the degree of any minimally required (useful) bypasses of energy-conserving reactions in the respiratory chain, and the magnitude of any 'leaks' in the inner mitochondrial membrane are key research needs.

Protein folding and unfolding: proline cis-trans isomerization at the c subunits of F1 FO -ATPase might open a high conductance ion channel.

The c subunits, which constitute the c-ring apparatus of the F1 FO -ATPase, could be the main components of the mitochondrial permeability transition pore (mPTP). The well-known modulator of the mPTP formation and opening is the cyclophilin D (CyPD), a peptidyl-prolyl cis-trans isomerase. On the loop, which connects the two hairpin α-helix of c subunit, is present the unique proline residue (Pro40 ) that could be a biological target of CyPD. Indeed, the proline cis-trans isomerization might provide the switch that interconverts the open/closed states of the pore by pulling out the c-ring lipid plug.

HrpB4 from Xanthomonas campestris pv. vesicatoria acts similarly to SctK proteins and promotes the docking of the predicted sorting platform to the type III secretion system.

The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper and tomato plants. Pathogenicity of X. campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates bacterial effector proteins into plant cells. At least nine membrane-associated and cytoplasmic components of the secretion apparatus are homologous to corresponding Sct (secretion and cellular translocation) proteins from animal pathogens, suggesting a similar structural organisation of T3S systems in different bacterial species. T3S in X. campestris pv. vesicatoria also depends on non-conserved proteins with yet unknown function including the essential pathogenicity factor HrpB4. Here, we show that HrpB4 localises to the cytoplasm and the bacterial membranes and interacts with the cytoplasmic domain of the inner membrane (IM) ring component HrcD and the cytoplasmic HrcQ protein. The analysis of HrpB4 deletion derivatives revealed that deletion of the N- or C-terminal protein region affects the interaction of HrpB4 with HrcQ and HrcD as well as its contribution to pathogenicity. HrcQ is a component of the predicted sorting platform, which was identified in animal pathogens as a dynamic heterooligomeric protein complex and associates with the IM ring via SctK proteins. HrcQ complex formation was previously shown by fluorescent microscopy analysis and depends on the presence of the T3S system. In the present study, we provide experimental evidence that the absence of HrpB4 severely affects the docking of HrcQ complexes to the T3S system but does not significantly interfere with HrcQ complex formation in the bacterial cytoplasm. Taken together, our data suggest that HrpB4 links the predicted cytoplasmic sorting platform to the IM rings of the T3S system.

Rotor subunits adaptations in ATP synthases from photosynthetic organisms.

Driven by transmembrane electrochemical ion gradients, F-type ATP synthases are the primary source of the universal energy currency, adenosine triphosphate (ATP), throughout all domains of life. The ATP synthase found in the thylakoid membranes of photosynthetic organisms has some unique features not present in other bacterial or mitochondrial systems. Among these is a larger-than-average transmembrane rotor ring and a redox-regulated switch capable of inhibiting ATP hydrolysis activity in the dark by uniquely adapted rotor subunit modifications. Here, we review recent insights into the structure and mechanism of ATP synthases specifically involved in photosynthesis and explore the cellular physiological consequences of these adaptations at short and long time scales.

HrpB7 from Xanthomonas campestris pv. vesicatoria is an essential component of the type III secretion system and shares features of HrpO/FliJ/YscO family members.

The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria translocates effector proteins via a type III secretion system (T3SS) into eukaryotic cells. The T3SS spans both bacterial membranes and consists of more than 20 proteins, 9 of which are conserved in plant and animal pathogens and constitute the core subunits of the secretion apparatus. T3S in X. campestris pv. vesicatoria also depends on nonconserved proteins with yet unknown function including HrpB7, which contains predicted N- and C-terminal coiled-coil regions. In the present study, we provide experimental evidence that HrpB7 forms stable oligomeric complexes. Interaction and localisation studies suggest that HrpB7 interacts with inner membrane and predicted cytoplasmic (C) ring components of the T3SS but is dispensable for the assembly of the C ring. Additional interaction partners of HrpB7 include the cytoplasmic adenosinetriphosphatase HrcN and the T3S chaperone HpaB. The interaction of HrpB7 with T3SS components as well as complex formation by HrpB7 depends on the presence of leucine heptad motifs, which are part of the predicted N- and C-terminal coiled-coil structures. Our data suggest that HrpB7 forms multimeric complexes that associate with the T3SS and might serve as a docking site for the general T3S chaperone HpaB.

Molecular architecture of the N-type ATPase rotor ring from Burkholderia pseudomallei.

The genome of the highly infectious bacterium Burkholderia pseudomallei harbors an atp operon that encodes an N-type rotary ATPase, in addition to an operon for a regular F-type rotary ATPase. The molecular architecture of N-type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N1No-type ATPase and investigated the structure and ion specificity of its membrane-embedded c-ring rotor by single-particle electron cryo-microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low-density, low-CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c-ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c17 ring is H+ specific, demonstrating that the ATPase is proton-coupled. The c17 ring stoichiometry results in a very high ion-to-ATP ratio of 5.7. We propose that this N-ATPase is a highly efficient proton pump that helps these melioidosis-causing bacteria to survive in the hostile, acidic environment of phagosomes.

Internal and external components of the bacterial flagellar motor rotate as a unit.

Most bacteria that swim, including Escherichia coli, are propelled by helical filaments, each driven at its base by a rotary motor powered by a proton or a sodium ion electrochemical gradient. Each motor contains a number of stator complexes, comprising 4MotA 2MotB or 4PomA 2PomB, proteins anchored to the rigid peptidoglycan layer of the cell wall. These proteins exert torque on a rotor that spans the inner membrane. A shaft connected to the rotor passes through the peptidoglycan and the outer membrane through bushings, the P and L rings, connecting to the filament by a flexible coupling known as the hook. Although the external components, the hook and the filament, are known to rotate, having been tethered to glass or marked by latex beads, the rotation of the internal components has remained only a reasonable assumption. Here, by using polarized light to bleach and probe an internal YFP-FliN fusion, we show that the innermost components of the cytoplasmic ring rotate at a rate similar to that of the hook.

Synthesis of C-ring-modified blebbistatin derivatives and evaluation of their myosin II ATPase inhibitory potency.

(S)-Blebbistatin is a micromolar myosin II ATPase inhibitor that is extensively used in research. In search of analogs with improved potency, we have synthesized for the first time C-ring modified analogs. We introduced hydroxymethyl or allyloxymethyl functionalities in search of additional favorable interactions and a more optimal filling of the binding pocket. Unfortunately, the resulting compounds did not significantly inhibit the ATPase activity of rabbit skeletal-muscle myosin II. This and earlier reports suggest that rational design of potent myosin II inhibitors based on the architecture of the blebbistatin binding pocket is an ineffective strategy.

MinD-like ATPase FlhG effects location and number of bacterial flagella during C-ring assembly.

The number and location of flagella, bacterial organelles of locomotion, are species specific and appear in regular patterns that represent one of the earliest taxonomic criteria in microbiology. However, the mechanisms that reproducibly establish these patterns during each round of cell division are poorly understood. FlhG (previously YlxH) is a major determinant for a variety of flagellation patterns. Here, we show that FlhG is a structural homolog of the ATPase MinD, which serves in cell-division site determination. Like MinD, FlhG forms homodimers that are dependent on ATP and lipids. It interacts with a complex of the flagellar C-ring proteins FliM and FliY (also FliN) in the Gram-positive, peritrichous-flagellated Bacillus subtilis and the Gram-negative, polar-flagellated Shewanella putrefaciens. FlhG interacts with FliM/FliY in a nucleotide-independent manner and activates FliM/FliY to assemble with the C-ring protein FliG in vitro. FlhG-driven assembly of the FliM/FliY/FliG complex is strongly enhanced by ATP and lipids. The protein shows a highly dynamic subcellular distribution between cytoplasm and flagellar basal bodies, suggesting that FlhG effects flagellar location and number during assembly of the C-ring. We describe the molecular evolution of a MinD-like ATPase into a flagellation pattern effector and suggest that the underappreciated structural diversity of the C-ring proteins might contribute to the formation of different flagellation patterns.

The c-Ring of the F1FO-ATP Synthase: Facts and Perspectives.

The F1FO-ATP synthase is the only enzyme in nature endowed with bi-functional catalytic mechanism of synthesis and hydrolysis of ATP. The enzyme functions, not only confined to energy transduction, are tied to three intrinsic features of the annular arrangement of c subunits which constitutes the so-called c-ring, the core of the membrane-embedded FO domain: (i) the c-ring constitution is linked to the number of ions (H(+) or Na(+)) channeled across the membrane during the dissipation of the transmembrane electrochemical gradient, which in turn determines the species-specific bioenergetic cost of ATP, the "molecular currency unit" of energy transfer in all living beings; (ii) the c-ring is increasingly involved in the mitochondrial permeability transition, an event linked to cell death and to most mitochondrial dysfunctions; (iii) the c subunit species-specific amino acid sequence and susceptibility to post-translational modifications can address antibacterial drug design according to the model of enzyme inhibitors which target the c subunits. Therefore, the simple c-ring structure not only allows the F1FO-ATP synthase to perform the two opposite tasks of molecular machine of cell life and death, but it also amplifies the enzyme's potential role as a drug target.

ATP synthases from archaea: the beauty of a molecular motor.

Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A1AO ATP synthases, a class of ATP synthases distinct from the F1FO ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V1VO ATPases of eukaryotes. A1AO ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A1AO ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A1AO ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H(+), Na(+) or even H(+) and Na(+) using enzymes. The evolution of the H(+) binding site to a Na(+) binding site and its implications for the energy metabolism and physiology of the cell are discussed.

Stress Corrosion Cracking of 316L Stainless Steel Additively Manufactured with Sinter-Based Material Extrusion.

This study investigates the stress corrosion cracking (SCC) behavior of type 316L stainless steel (SS316L) produced with sinter-based material extrusion additive manufacturing (AM). Sinter-based material extrusion AM produces SS316L with microstructures and mechanical properties comparable to its wrought counterpart in the annealed condition. However, despite extensive research on SCC of SS316L, little is known about the SCC of sinter-based AM SS316L. This study focuses on the influence of sintered microstructures on SCC initiation and crack-branching susceptibility. Custom-made C-rings were exposed to different stress levels in acidic chloride solutions at various temperatures. Solution-annealed (SA) and cold-drawn (CD) wrought SS316L were also tested to understand the SCC behavior of SS316L better. Results showed that sinter-based AM SS316L was more susceptible to SCC initiation than SA wrought SS316L but more resistant than CD wrought SS316L, as determined by the crack initiation time. Sinter-based AM SS316L showed a noticeably lower tendency for crack-branching than both wrought SS316L counterparts. The investigation was supported by comprehensive pre- and post-test microanalysis using light optical microscopy, scanning electron microscopy, electron backscatter diffraction, and micro-computed tomography.

Oxidation and degradation of (epi)gallocatechin gallate (EGCG/GCG) and (epi)catechin gallate (ECG/CG) in alkali solution.

The oxidative decomposition/degradation of two main tea flavanols, EGCG/GCG and ECG/CG, was studied in alkaline solution under ultrasonic-assisted thermal conditions. The study employed HPLC-ESI-ToF-MS to identify the products generated by atmospheric oxygen oxidation and various base-catalyzed reactions. Strong basic condition led to accelerated hydrolysis and oxidation of EGCG/GCG and ECG/CG and yielded gallic acid, de-galloyl flavanols and corresponding o-quinone derivatives. Meanwhile, peroxidation or base-catalyzed cleavage and rearrangement occurred extensively on C- and B-rings of flavanol and generated various simpler aldehydes or acids. Besides, a number of dimers/trimers were produced. This contribution provides empirical proof of oxidative degradation of flavanols under strong alkaline condition. Meanwhile, detailed reaction mechanisms of C-/B-ring degradation and dimerization/polymerization phenomena are proposed to help understand the structural changes of flavanols under strong alkaline conditions.

Strategies for elucidation of the structure and function of the large membrane protein complex, FoF1-ATP synthase, by nuclear magnetic resonance.

Nuclear magnetic resonance (NMR) investigation of large membrane proteins requires well-focused questions and critical techniques. Here, research strategies for FoF1-ATP synthase, a membrane-embedded molecular motor, are reviewed, focusing on the ß-subunit of F1-ATPase and c-subunit ring of the enzyme. Segmental isotope-labeling provided 89% assignment of the main chain NMR signals of thermophilic Bacillus (T)F1ß-monomer. Upon nucleotide binding to Lys164, Asp252 was shown to switch its hydrogen-bonding partner from Lys164 to Thr165, inducing an open-to-closed bend motion of TF1ß-subunit. This drives the rotational catalysis. The c-ring structure determined by solid-state NMR showed that cGlu56 and cAsn23 of the active site took a hydrogen-bonded closed conformation in membranes. In 505 kDa TFoF1, the specifically isotope-labeled cGlu56 and cAsn23 provided well-resolved NMR signals, which revealed that 87% of the residue pairs took a deprotonated open conformation at the Foa-c subunit interface, whereas they were in the closed conformation in the lipid-enclosed region.

Novel Catabolic Pathway of Quercetin-3-O-Rutinose-7-O-α-L-Rhamnoside by Lactobacillus plantarum GDMCC 1.140: The Direct Fission of C-Ring.

Lychee pulp phenolics (LPP) is mainly catabolized in the host colon, increasing the abundances of Bacteroides and Lactobacillus. Herein, five selected gut microbial strains (Bacteroides uniformis, B. thetaiotaomicron, Lactobacillus rhamnosus, L. plantarum, and L. acidophilus) were separately incubated with LPP to ascertain the specific strains participating in phenolic metabolism and the corresponding metabolites. The results indicated that B. uniformis, L. rhamnosus, and L. plantarum were involved in LPP utilization, contributing to 52.37, 28.33, and 45.11% of LPP degradation after 48 h fermentation, respectively. Unprecedentedly, the metabolic pathway of the major phenolic compound quercetin-3-O-rutinose-7-O-α-L-rhamnoside by L. plantarum, appeared to be the direct fission of C-ring at C2-O1 and C3-C4 bonds, which was proved from the occurrence of two substances with the deprotonated molecule [M-H]- ion at m/z 299 and 459, respectively. Meanwhile, it was fully confirmed that B. uniformis participated in the catabolism of isorhamnetin glycoside and procyanidin B2. In the B. uniformis culture, kaempferol was synthesized through dehydroxylation of quercetin which could be catabolized into alphitonin by L. rhamnosus. Furthermore, LPP metabolites exerted higher antioxidant activity than their precursors and gave clues to understand the interindividual differences for phenolic metabolism by gut microbiota.

Cooperation among c-subunits of FoF1-ATP synthase in rotation-coupled proton translocation.

In FoF1-ATP synthase, proton translocation through Fo drives rotation of the c-subunit oligomeric ring relative to the a-subunit. Recent studies suggest that in each step of the rotation, key glutamic acid residues in different c-subunits contribute to proton release to and proton uptake from the a-subunit. However, no studies have demonstrated cooperativity among c-subunits toward FoF1-ATP synthase activity. Here, we addressed this using Bacillus PS3 ATP synthase harboring a c-ring with various combinations of wild-type and cE56D, enabled by genetically fused single-chain c-ring. ATP synthesis and proton pump activities were decreased by a single cE56D mutation and further decreased by double cE56D mutations. Moreover, activity further decreased as the two mutation sites were separated, indicating cooperation among c-subunits. Similar results were obtained for proton transfer-coupled molecular simulations. The simulations revealed that prolonged proton uptake in mutated c-subunits is shared between two c-subunits, explaining the cooperation observed in biochemical assays.

Cells need to be able to store and transfer energy to fuel their various activities. To do this, they produce a small molecule called ATP to carry the energy, which is then released when the ATP is broken down. An enzyme found in plants, animals and bacteria, called F_oF₁ ATP synthase, can both create and use ATP. When it does this, protons, or positive hydrogen ions, are transported across cellular boundaries called membranes. The region of the enzyme that is responsible for pumping the protons contains different parts known as the c-ring and the a-subunit. The movement of protons drives the c-ring to rotate relative to the a-subunit, which leads to producing ATP. Previous research using simulations and the protein structures found there are two or three neighbouring amino acids in the c-ring that face the a-subunit, suggesting that these amino acids act together to drive the rotation. To test this hypothesis, Mitome et al. mutated these amino acids to examine the effect on the enzyme's ability to produce ATP. A single mutation reduced the production of ATP, which decreased even further with mutations in two of the amino acids. The extent of this decrease depended on the distance between the two mutations in the c-ring. Simulations of these changes also found similar results. This indicates there is coordination between different parts of the c-ring to increase the rate of ATP production. This study offers new insights into the molecular processes controlling ATP synthesis and confirms previous theoretical research. This will interest specialists in bioenergetics because it addresses a fundamental biological question with broad impact.

Ring assembly of c subunits of F0 F1 -ATP synthase in Propionigenium modestum requires YidC and UncI following MPIase-dependent membrane insertion.

The c subunits of F0 F1 -ATP synthase (F0 c) assemble into a ring structure, following membrane insertion that is dependent on both glycolipid MPIase and protein YidC. We analyzed the insertion and assembly processes of Propionigenium modestum F0 c (Pm-F0 c), of which the ring structure is resistant to SDS. Ring assembly of Pm-F0 c requires P. modestum UncI (Pm-UncI). Ring assembly of in vitro synthesized Pm-F0 c was observed when both YidC and Pm-UncI were reconstituted into liposomes of Escherichia coli phospholipids. Under the physiological conditions where spontaneous insertion had been blocked by diacylglycerol, MPIase was necessary for Pm-F0 c insertion allowing the subsequent YidC/Pm-UncI-dependent ring assembly. Thus, we have succeeded in the complete reconstitution of membrane insertion and subsequent ring assembly of Pm-F0 c.