Heat treatment of thioredoxin fusions increases the purity of α-helical transmembrane protein constructs.

Membrane proteins play key roles in cellular signaling and transport, represent the majority of drug targets, and are implicated in many diseases. Their relevance renders them important subjects for structural, biophysical, and functional investigations. However, obtaining membrane proteins in high purities is often challenging with conventional purification steps alone. To address this issue, we present here an approach to increase the purity of α-helical transmembrane proteins. Our approach exploits the Thioredoxin (Trx) tag system, which is able to confer some of its favorable properties, such as high solubility and thermostability, to its fusion partners. Using Trx fusions of transmembrane helical hairpin constructs derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) and a bacterial ATP synthase, we establish conditions for the successful implementation of the selective heat treatment procedure to increase sample purity. We further examine systematically its efficacy with respect to different incubation times and temperatures using quantitative gel electrophoresis. We find that minute-timescale heat treatment of Trx-tagged fusion constructs with temperatures ranging from 50 to 90°C increases the purity of the membrane protein samples from ~60 to 98% even after affinity purification. We show that this single-step approach is even applicable in cases where regular selective heat purification from crude extracts, as reported for Trx fusions to soluble proteins, fails. Overall, our approach is easy to integrate into existing purification strategies and provides a facile route for increasing the purity of membrane protein constructs after purification by standard chromatography approaches.

Conserved role of ATP synthase in mammalian cilia.

We previously found that ATP synthases localize to male-specific sensory cilia and control the ciliary response by regulating polycystin signalling in Caenorhabditis elegans. Herein, we discovered that the ciliary localization of ATP synthase is evolutionarily conserved in mammals. We showed that the ATP synthase subunit F1ß is colocalized with the cilia marker acetylated α-tubulin in both mammalian renal epithelial cells (MDCK) and normal mouse cholangiocytes (NMCs). Treatment with ATP synthase inhibitor oligomycin impaired ciliogenesis in MDCK cells, and F1ß was co-immunoprecipitated with PKD2 in mammalian cells. Our study provides evidence for the evolutionarily conserved localization of ATP synthase in cilia from worm to mammals. Defects in ATP synthase can lead to ciliary dysfunction, which may be a potential mechanism of polycystic kidney disease.

Structure of mycobacterial ATP synthase bound to the tuberculosis drug bedaquiline.

Tuberculosis-the world's leading cause of death by infectious disease-is increasingly resistant to current first-line antibiotics1. The bacterium Mycobacterium tuberculosis (which causes tuberculosis) can survive low-energy conditions, allowing infections to remain dormant and decreasing their susceptibility to many antibiotics2. Bedaquiline was developed in 2005 from a lead compound identified in a phenotypic screen against Mycobacterium smegmatis3. This drug can sterilize even latent M. tuberculosis infections4 and has become a cornerstone of treatment for multidrug-resistant and extensively drug-resistant tuberculosis1,5,6. Bedaquiline targets the mycobacterial ATP synthase3, which is an essential enzyme in the obligate aerobic Mycobacterium genus3,7, but how it binds the intact enzyme is unknown. Here we determined cryo-electron microscopy structures of M. smegmatis ATP synthase alone and in complex with bedaquiline. The drug-free structure suggests that hook-like extensions from the α-subunits prevent the enzyme from running in reverse, inhibiting ATP hydrolysis and preserving energy in hypoxic conditions. Bedaquiline binding induces large conformational changes in the ATP synthase, creating tight binding pockets at the interface of subunits a and c that explain the potency of this drug as an antibiotic for tuberculosis.

Structural Evolution of the Glacier Ice Worm Fo ATP Synthase Complex.

The segmented annelid worm, Mesenchytraeus solifugus, is a permanent resident of temperate, maritime glaciers in the Pacific northwestern region of North America, displaying atypically high intracellular ATP levels which have been linked to its unusual ability to thrive in hydrated glacier ice. We have shown previously that ice worms contain a highly basic, carboxy terminal extension on their ATP6 regulatory subunit, likely acquired by horizontal gene transfer from a microbial dietary source. Here we examine the full complement of F1F0 ATP synthase structural subunits with attention to non-conservative, ice worm-specific structural modifications. Our genomics analyses and molecular models identify putative proton shuttling domains on either side of the F0 hemichannel, which predictably function to enhance proton flow across the mitochondrial membrane. Other components of the ice worm ATP synthase complex have remained largely unchanged in the context of Metazoan evolution.

A Na+ A1 AO ATP synthase with a V-type c subunit in a mesophilic bacterium.

A1 AO ATP synthases with a V-type c subunit have only been found in hyperthermophilic archaea which makes bioenergetic analyses impossible due to the instability of liposomes at high temperatures. A search for a potential archaeal A1 AO ATP synthase with a V-type c subunit in a mesophilic organism revealed an A1 AO ATP synthase cluster in the anaerobic, acetogenic bacterium Eubacterium limosum KIST612. The enzyme was purified to apparent homogeneity from cells grown on methanol to a specific activity of 1.2 U·mg-1 with a yield of 12%. The enzyme contained subunits A, B, C, D, E, F, H, a, and c. Subunit c is predicted to be a typical V-type c subunit with only one ion (Na+ )-binding site. Indeed, ATP hydrolysis was strictly Na+ -dependent. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis, but inhibition was relieved by addition of Na+ . Na+ was shown directly to abolish binding of the fluorescence DCCD derivative, NCD-4, to subunit c, demonstrating a competition of Na+ and DCCD/NCD-4 for a common binding site. After incorporation of the A1 AO ATP synthase into liposomes, ATP-dependent primary transport of 22 Na+ as well as ΔµNa+ -driven ATP synthesis could be demonstrated. The Na+ A1 AO ATP synthase from E. limosum is the first ATP synthase with a V-type c subunit from a mesophilic organism. This will enable future bioenergetic analysis of these unique ATP synthases.

ATP Synthase: Structure, Function and Inhibition.

Oxidative phosphorylation is carried out by five complexes, which are the sites for electron transport and ATP synthesis. Among those, Complex V (also known as the F1F0 ATP Synthase or ATPase) is responsible for the generation of ATP through phosphorylation of ADP by using electrochemical energy generated by proton gradient across the inner membrane of mitochondria. A multi subunit structure that works like a pump functions along the proton gradient across the membranes which not only results in ATP synthesis and breakdown, but also facilitates electron transport. Since ATP is the major energy currency in all living cells, its synthesis and function have widely been studied over the last few decades uncovering several aspects of ATP synthase. This review intends to summarize the structure, function and inhibition of the ATP synthase.

The structure of the catalytic domain of the ATP synthase from Mycobacterium smegmatis is a target for developing antitubercular drugs.

The crystal structure of the F1-catalytic domain of the adenosine triphosphate (ATP) synthase has been determined from Mycobacterium smegmatis which hydrolyzes ATP very poorly. The structure of the α3ß3-component of the catalytic domain is similar to those in active F1-ATPases in Escherichia coli and Geobacillus stearothermophilus However, its ε-subunit differs from those in these two active bacterial F1-ATPases as an ATP molecule is not bound to the two α-helices forming its C-terminal domain, probably because they are shorter than those in active enzymes and they lack an amino acid that contributes to the ATP binding site in active enzymes. In E. coli and G. stearothermophilus, the α-helices adopt an "up" state where the α-helices enter the α3ß3-domain and prevent the rotor from turning. The mycobacterial F1-ATPase is most similar to the F1-ATPase from Caldalkalibacillus thermarum, which also hydrolyzes ATP poorly. The ßE-subunits in both enzymes are in the usual "open" conformation but appear to be occupied uniquely by the combination of an adenosine 5'-diphosphate molecule with no magnesium ion plus phosphate. This occupation is consistent with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic products are probably the basis of the inhibition of ATP hydrolysis. It can be envisaged that specific as yet unidentified small molecules might bind to the F1 domain in Mycobacterium tuberculosis, prevent ATP synthesis, and inhibit the growth of the pathogen.

Purification of a Crenarchaeal ATP Synthase in the Light of the Unique Bioenergetics of Ignicoccus Species.

In this study, the ATP synthase of Ignicoccus hospitalis was purified, characterized, and structurally compared to the respective enzymes of the other Ignicoccus species, to shed light on energy conservation in this unique group of archaea. The crenarchaeal genus Ignicoccus comprises three described species, i.e., I. hospitalis and Ignicoccus islandicus from hot marine sediments near Iceland and Ignicoccus pacificus from a hydrothermal vent system in the Pacific Ocean. This genus is unique among all archaea due to the unusual cell envelope, consisting of two membranes that enclose a large intermembrane compartment (IMC). I. hospitalis is the best studied member of this genus, mainly because it is the only known host for the potentially parasitic archaeon Nanoarchaeum equitansI. hospitalis grows chemolithoautotrophically, and its sole energy-yielding reaction is the reduction of elemental sulfur with molecular hydrogen, forming large amounts of hydrogen sulfide. This reaction generates an electrochemical gradient, which is used by the ATP synthase, located in the outer cellular membrane, to generate ATP inside the IMC. The genome of I. hospitalis encodes nine subunits of an A-type ATP synthase, which we could identify in the purified complex. Although the maximal in vitro activity of the I. hospitalis enzyme was measured around pH 6, the optimal stability of the A1AO complex seemed to be at pH 9. Interestingly, the soluble A1 subcomplexes of the different Ignicoccus species exhibited significant differences in their apparent molecular masses in native electrophoresis, although their behaviors in gel filtration and chromatography-mass spectrometry were very similar.IMPORTANCE The Crenarchaeota represent one of the major phyla within the Archaea domain. This study describes the successful purification of a crenarchaeal ATP synthase. To date, all information about A-type ATP synthases is from euryarchaeal enzymes. The fact that it has not been possible to purify this enzyme complex from a member of the Crenarchaeota until now points to significant differences in stability, possibly caused by structural alterations. Furthermore, the study subject I. hospitalis has a particular importance among crenarchaeotes, since it is the only known host of N. equitans The energy metabolism in this system is still poorly understood, and our results can help elucidate the unique relationship between these two microbes.

Cryo-EM of ATP synthases.

ATP synthases are rotary enzymes found in bacteria, chloroplasts, and mitochondria. These complexes produce the majority of cellular ATP in aerobic cells using energy from the transmembrane proton motive force established by the electron transport chain. In mitochondria, dimeric ATP synthase is essential for formation of the inner membrane cristae. While rotary catalysis in the soluble F1 region has been studied extensively by X-ray crystallography, the structure of the membrane embedded FO region remained elusive until recently. In the past few years, electron cryomicroscopy structures of mitochondrial, chloroplast, and bacterial ATP synthases have revealed the architecture of the FO region, helping to explain the mechanisms of proton translocation, dimerization of the enzyme in mitochondria, and cristae formation. These structures also show that ATP synthases exist in different conformational states, illustrating the flexibility and dynamics of the complex.

The Broad Aryl Acid Specificity of the Amide Bond Synthetase McbA Suggests Potential for the Biocatalytic Synthesis of Amides.

Amide bond formation is one of the most important reactions in pharmaceutical synthetic chemistry. The development of sustainable methods for amide bond formation, including those that are catalyzed by enzymes, is therefore of significant interest. The ATP-dependent amide bond synthetase (ABS) enzyme McbA, from Marinactinospora thermotolerans, catalyzes the formation of amides as part of the biosynthetic pathway towards the marinacarboline secondary metabolites. The reaction proceeds via an adenylate intermediate, with both adenylation and amidation steps catalyzed within one active site. In this study, McbA was applied to the synthesis of pharmaceutical-type amides from a range of aryl carboxylic acids with partner amines provided at 1-5 molar equivalents. The structure of McbA revealed the structural determinants of aryl acid substrate tolerance and differences in conformation associated with the two half reactions catalyzed. The catalytic performance of McbA, coupled with the structure, suggest that this and other ABS enzymes may be engineered for applications in the sustainable synthesis of pharmaceutically relevant (chiral) amides.

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales, Methanosarcinales, and Methanococcales, as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales.

Conformational dynamics of the rotary subunit F in the A3 B3 DF complex of Methanosarcina mazei Gö1 A-ATP synthase monitored by single-molecule FRET.

In archaea the A1 AO ATP synthase uses a transmembrane electrochemical potential to generate ATP, while the soluble A1 domain (subunits A3 B3 DF) alone can hydrolyse ATP. The three nucleotide-binding AB pairs form a barrel-like structure with a central orifice that hosts the rotating central stalk subunits DF. ATP binding, hydrolysis and product release cause a conformational change inside the A:B-interface, which enforces the rotation of subunits DF. Recently, we reported that subunit F is a stimulator of ATPase activity. Here, we investigated the nucleotide-dependent conformational changes of subunit F relative to subunit D during ATP hydrolysis in the A3 B3 DF complex of the Methanosarcina mazei Gö1 A-ATP synthase using single-molecule Förster resonance energy transfer. We found two conformations for subunit F during ATP hydrolysis.

Delivery of membrane proteins into small and giant unilamellar vesicles by charge-mediated fusion.

One of the current challenges in synthetic biology is the production of stable membrane mimetic systems and the insertion of components in these systems. Here, we employ fusion of oppositely charged liposomes to deliver separately reconstituted membrane proteins into a common lipid bilayer. After a systematic evaluation of different lipid compositions by lipid mixing and size distribution analysis, suitable conditions were further investigated for proteoliposome fusion. With this technique, we functionally coreconstituted bo3 oxidase and ATP synthase from Escherichia coli into unilamellar liposomes ranging from 100 nm to 50 µm in size. The presented method is a simple and versatile tool for oriented membrane protein reconstitution to produce biomimetic systems with increased complexity.

Studies of the viral binding proteins of shrimp BP53, a receptor of white spot syndrome virus.

The specific binding between viral attachment proteins (VAPs) of a virus and its cellular receptors on host cells mediates virus entry into host cells, which triggers subsequent viral infections. Previous studies indicate that F1 ATP synthase ß subunit (named BP53), is found on the surface of shrimp cells and involved in white spot syndrome virus (WSSV) infection by functioning as a potential viral receptor. Herein, in a far-western blotting assay, three WSSV proteins with molecular weights of 28 kDa, 37 kDa, and >50 kDa were found to interact with BP53. The 28 kDa and 37 kDa proteins were identified as the envelope protein VP28 and VP37 of WSSV respectively, which could be recognized by the polyclonal antibodies. Enzyme-linked immunosorbent binding assays revealed that VP37 contributed to almost 80% of the binding capability for BP53 compared with the same amount of total WSSV protein. The relationship between BP53 and its complementary interacting protein, VP37, was visualized using a co-localization assay. Bound VP37 on the cell surface co-localized with BP53 and shared a similar subcellular location on the outer surface of shrimp cells. Pearson's correlation coefficients reached to 0.67 ± 0.05 and the Mander's overlap coefficients reached 0.70 ± 0.05, which indicated a strong relationship between the localization of BP53 and bound rVP37. This provides evidence for an interaction between BP53 and VP37 obtained at the molecular and cellular levels, supporting the hypothesis that BP53 serves as a receptor for WSSV by binding to VP37. The identification of the viral binding proteins of shrimp BP53 is helpful for better understanding the pathogenic mechanisms of WSSV to infect shrimp at the cellular level.

The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase.

A1AO ATP synthases couple ion-transport of the AO sector and ATP synthesis/hydrolysis of the A3B3-headpiece via their stalk subunits D and F. Here, we produced and purified stable A3B3D- and A3B3DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Gö1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates (Vmax) of A3B3D and A3B3DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a Vmax of 7.9 s(-1) and 30.4 s(-1), respectively, while the KM is the same for both. Deletions of the N- or C-termini of subunit F abolished the effect of ATP hydrolysis activation. We generated subunit F mutant proteins with single amino acid substitutions and demonstrated that the subunit F residues S84 and R88 are important in stimulating ATP hydrolysis. Hybrid formation of the A3B3D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit ε of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis.

Computational modeling of TC0583 as a putative component of the Chlamydia muridarum V-type ATP synthase complex and assessment of its protective capabilities as a vaccine antigen.

Numerous Chlamydia trachomatis proteins have been identified as potential subunit vaccines, of which the major outer-membrane protein (MOMP) has, so far, proven the most efficacious. Recently, subunit A of the V-type ATP synthase (ATPase; TC0582) complex was shown to elicit partial protection against infection. Computational modeling of a neighboring gene revealed a novel subunit of the V-type ATPase (TC0583). To determine if this newly identified subunit could induce protection and/or enhance the partial protection provided by subunit A alone, challenge studies were performed using a combination of these recombinant proteins. The TC0583 subunit alone and concurrently with TC0582, was used to vaccinate BALB/c mice utilizing CpG-1826 and Montanide ISA 720 VG as adjuvants. Vaccinated animals were challenged intranasally with Chlamydia muridarum and the course of the infection was followed. Mice immunized with individual antigens showed minimal alleviation of body weight reduction; however, mice immunized with TC0583 and TC0582 in combination, displayed weight loss levels close to those observed with MOMP. Importantly, immunization with a combination of recombinant subunit proteins reduced chlamydial inclusion forming units by approximately a log-fold. These protection levels support that, these highly conserved Chlamydia proteins, in combination with other antigens, may serve as potential vaccine candidates.

Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans.

ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase.

FoF1-ATP synthase of Streptomyces fradiae ATCC 19609: structural, biochemical, and functional characterization.

The patterns of protein phosphorylation in inverted membrane vesicles from the strain Streptomyces fradiae ATCC 19609 were investigated to elucidate the mechanisms of regulation of bacterial membrane bound FoF1-ATP synthase. We found for the first time by two-dimensional gel electrophoresis and mass spectrometry that the ß- and b-subunits of the FoF1-ATP synthase complex undergo phosphorylation; 20 proteins with known functions were identified. All eight subunits of FoF1-ATP synthase, i.e. α, ß, γ, δ, ε, a, b, and c, were cloned into Escherichia coli and expressed as recombinant proteins. Using a crude preparation of serine/threonine protein kinases, we demonstrated the phosphorylation of recombinant γ-, ß-, α- and ε-subunits. The ß-subunit was phosphorylated both as a recombinant protein and in vesicles. Differential phosphorylation of membrane-bound and recombinant proteins can be attributed to different pools of protein kinases in each preparation; in addition, certain steps of FoF1-ATP synthase assembly and function might be accompanied by individual phosphorylation patterns. The structure of the operon containing all subunits and regulatory protein I was identified. The phylogenetic similarity of FoF1-ATP synthase from Streptomyces fradiae ATCC 19609 with the respective proteins in saprophytic and pathogenic (including Mycobacterium tuberculosis) bacteria was investigated. Thus, bacterial serine/threonine protein kinases are important for the regulation of FoF1-ATP synthase. From the practical standpoint, our results provide a basis for designing targeted antibacterial drugs.

Opposite rotation directions in the synthesis and hydrolysis of ATP by the ATP synthase: hints from a subunit asymmetry.

The ATP synthase can be imagined as a reversible H(+)-translocating channel embedded in the membrane, FO portion, coupled to a protruding catalytic portion, F1. Under physiological conditions the F1FO complex synthesizes ATP by exploiting the transmembrane electrochemical gradient of protons and their downhill movement. Alternatively, under other patho-physiological conditions it exploits ATP hydrolysis to energize the membrane by uphill pumping protons. The reversibility of the mechanism is guaranteed by the structural coupling between the hydrophilic F1 and the hydrophobic FO. Which of the two opposite processes wins in the energy-transducing membrane complex depends on the thermodynamic balance between the protonmotive force (Δp) and the phosphorylation potential of ATP (ΔG P). Accordingly, while Δp prevalence drives ATP synthesis by translocating protons from the membrane P-side to the N-side and generating anticlockwise torque rotation (viewed from the matrix), ΔG P drives ATP hydrolysis by chemomechanical coupling of FO to F1 with clockwise torque. The direction of rotation is the same in all the ATP synthases, due to the conserved steric arrangement of the chiral a subunit of FO. The ability of this coupled bi-functional complex to produce opposite rotations in ATP synthesis and hydrolysis is explained on the basis of the a subunit asymmetry.