High Affinity ATP Research Articles

Structural basis of peptide secretion for Quorum sensing by ComA

Quorum sensing (QS) is a crucial regulatory mechanism controlling bacterial signalling and holds promise for novel therapies against antimicrobial resistance. In Gram-positive bacteria, such as Streptococcus pneumoniae, ComA is a conserved efflux pump responsible for the maturation and secretion of peptide signals, including the competence-stimulating peptide (CSP), yet its structure and function remain unclear. Here, we functionally characterize ComA as an ABC transporter with high ATP affinity and determined its cryo-EM structures in the presence or absence of CSP or nucleotides. Our findings reveal a network of strong electrostatic interactions unique to ComA at the intracellular gate, a putative binding pocket for two CSP molecules, and negatively charged residues facilitating CSP translocation. Mutations of these residues affect ComA’s peptidase activity in-vitro and prevent CSP export in-vivo. We demonstrate that ATP-Mg2+ triggers the outward-facing conformation of ComA for CSP release, rather than ATP alone. Our study provides molecular insights into the QS signal peptide secretion, highlighting potential targets for QS-targeting drugs.

Microwave‐Assisted Syntheses of Rhodamine, Rhodol, and Fluorescein Derivatives

AbstractA series of rhodol‐; fluorescein‐ and rhodamines‐based spirolactam compounds, bearing electron donor amines have been prepared. For this purpose we have redesigned the synthesis of the rhodol scaffold using 2‐(2,4‐dihydroxybenzoyl)benzoic acid obtaining one example rhodol methyl ester in good yields (25–30 %) Thus, one set of non‐cytotoxic rhodamine‐based compounds has been prepared using thermal and microwave assisted synthesis (40–78 %) and tested as high affinity ATP chemo‐sensors.

Metabolic adjustments in response to ATP spilling by the small DX protein in a Streptomyces strain.

ATP wasting is recognized as an efficient strategy to enhance metabolic activity and productivity of specific metabolites in several microorganisms. However, such strategy has been rarely implemented in Streptomyces species whereas antibiotic production by members of this genus is known to be triggered in condition of phosphate limitation that is correlated with a low ATP content. In consequence, to assess the effects of ATP spilling on the primary and specialized metabolisms of Streptomyces, the gene encoding the small synthetic protein DX, that has high affinity for ATP and dephosphorylates ATP into ADP, was cloned in the integrative vector pOSV10 under the control of the strong ErmE promoter. This construct and the empty vector were introduced into the species Streptomyces albogriseolus/viridodiastaticus yielding A37 and A36, respectively. A37 yielded higher biomass than A36 indicating that the DX-mediated ATP degradation resulted into a stimulation of A37 metabolism, consistently with what was reported in other microorganisms. The comparative analysis of the metabolomes of A36 and A37 revealed that A37 had a lower content in glycolytic and Tricarboxylic Acid Cycle intermediates as well as in amino acids than A36, these metabolites being consumed for biomass generation in A37. In contrast, the abundance of other molecules indicative either of energetic stress (ADP, AMP, UMP, ornithine and thymine), of activation (NAD and threonic acid) or inhibition (citramalic acid, fatty acids, TAG and L-alanine) of the oxidative metabolism, was higher in A37 than in A36. Furthermore, hydroxyl-pyrimidine derivatives and polycyclic aromatic polyketide antibiotics belonging to the angucycline class and thought to have a negative impact on respiration were also more abundantly produced by A37 than by A36. This comparative analysis thus revealed the occurrence in A37 of antagonistic metabolic strategies, namely, activation or slowing down of oxidative metabolism and respiration, to maintain the cellular energetic balance. This study thus demonstrated that DX constitutes an efficient biotechnological tool to enhance the expression of the specialized metabolic pathways present in the Streptomyces genomes that may include cryptic pathways. Its use thus might lead to the discovery of novel bioactive molecules potentially useful to human health.

Distinctive in vitro ATP Hydrolysis Activity of AtVIPP1, a Chloroplastic ESCRT-III Superfamily Protein in Arabidopsis.

Vesicle-inducing protein in plastid 1 (VIPP1), characteristic to oxygenic photosynthetic organisms, is a membrane-remodeling factor that forms homo-oligomers and functions in thylakoid membrane formation and maintenance. The cyanobacterial VIPP1 structure revealed a monomeric folding pattern similar to that of endosomal sorting complex required for transport (ESCRT) III. Characteristic to VIPP1, however, is its own GTP and ATP hydrolytic activity without canonical domains. In this study, we found that histidine-tagged Arabidopsis VIPP1 (AtVIPP1) hydrolyzed GTP and ATP to produce GDP and ADP in vitro, respectively. Unexpectedly, the observed GTPase and ATPase activities were biochemically distinguishable, because the ATPase was optimized for alkaline conditions and dependent on Ca2+ as well as Mg2+, with a higher affinity for ATP than GTP. We found that a version of AtVIPP1 protein with a mutation in its nucleotide-binding site, as deduced from the cyanobacterial structure, retained its hydrolytic activity, suggesting that Arabidopsis and cyanobacterial VIPP1s have different properties. Negative staining particle analysis showed that AtVIPP1 formed particle or rod structures that differed from those of cyanobacteria and Chlamydomonas. These results suggested that the nucleotide hydrolytic activity and oligomer formation of VIPP1 are common in photosynthetic organisms, whereas their properties differ among species.

Changes in hemoglobin function and isoform expression during embryonic development in the American alligator, Alligator mississippiensis.

In the developing embryos of egg-laying vertebrates, O2 flux takes place across a fixed surface area of the eggshell and the chorioallantoic membrane. In the case of crocodilians, the developing embryo may experience a decrease in O2 flux when the nest becomes hypoxic, which may cause compensatory adjustments in blood O2 transport. However, whether the switch from embryonic to adult hemoglobin isoforms (isoHbs) plays some role in these adjustments is unknown. Here, we provide a detailed characterization of the developmental switch of isoHb synthesis in the American alligator, Alligator mississippiensis. We examined the in vitro functional properties and subunit composition of purified alligator isoHbs expressed during embryonic developmental stages in normoxia and hypoxia (10% O2). We found distinct patterns of isoHb expression in alligator embryos at different stages of development, but these patterns were not affected by hypoxia. Specifically, alligator embryos expressed two main isoHbs: HbI, prevalent at early developmental stages, with a high O2 affinity and high ATP sensitivity, and HbII, prevalent at later stages and identical to the adult protein, with a low O2 affinity and high CO2 sensitivity. These results indicate that whole blood O2 affinity is mainly regulated by ATP in the early embryo and by CO2 and bicarbonate from the late embryo until adult life, but the developmental regulation of isoHb expression is not affected by hypoxia exposure.

Functional stability of CFTR depends on tight binding of ATP at its degenerate ATP-binding site.

Opening of the cystic fibrosis transmembrane conductance regulator (CFTR) channel is coupled to the motion of its two nucleotide-binding domains: they form a heterodimer sandwiching two functionally distinct ATP-binding sites (sites 1 and 2). While active ATP hydrolysis in site 2 triggers rapid channel closure, the functional role of stable ATP binding in the catalysis-incompetent (or degenerate) site 1, a feature conserved in many other ATP-binding cassette (ABC) transporter proteins, remains elusive. Here, we found that CFTR loses its prompt responsiveness to ATP after the channel is devoid of ATP for tens to hundreds of seconds. Mutants with weakened ATP binding in site 1 and the most prevalent disease-causing mutation, F508del, are more vulnerable to ATP depletion. In contrast, strengthening ligand binding in site 1 with N6 -(2-phenylethyl)-ATP, a high-affinity ATP analogue, or abolishing ATP hydrolysis in site 2 by the mutation D1370N, helps sustain a durable function of the otherwise unstable mutant channels. Thus, tight binding of ATP in the degenerate ATP-binding site is crucial to the functional stability of CFTR. Small molecules targeting site 1 may bear therapeutic potential to overcome the membrane instability of F508del-CFTR. KEY POINTS: During evolution, many ATP-binding cassette transporters - including the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, whose dysfunction causes cystic fibrosis (CF) - lose the ability to hydrolyse ATP in one of the two ATP-binding sites. Here we show that tight ATP binding at this degenerate site in CFTR is central for maintaining the stable, robust function of normal CFTR. We also demonstrate that membrane instability of the most common CF-causing mutant, F508del-CFTR, can be rescued by strengthening ATP binding at CFTR's degenerate site. Our data thus explain an evolutionary puzzle and offer a potential therapeutic strategy for CF.

Optimizing a biosensor‐based assay for ubiquitination activation

Ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) are proteins that perform post-translational modifications on protein substrates which promote essential functions such as inflammatory response, DNA repair, and protein degradation. Three transfer enzymes catalyze the ubiquitination pathway: E1, E2, and E3 which activate, conjugate, and ligate the Ub to its target protein, respectively. Despite the identification of these enzymes’ structure and function, a complete understanding of their catalytic mechanisms remains unclear. Elucidation of the ubiquitination mechanism could serve strategies for cancer treatment and other medicinal targets due to the degradation and activity-altering aspect of Ubls towards deregulated or misfolded proteins. Available assays for ubiquitination include gel-based assays, which do not give quantitative data in real time, the luciferase assay which can become costly, as well as assays that use radiotracers. We are trying to develop a continuous, relatively cheap, non-radioactive assay which can make ubiquitination research more accessible. Our approach uses fluorescent biosensors to monitor ATP hydrolysis of E1 in the ubiquitination pathway in order to measure enzyme activity. The ultimate goal is to couple this reaction to E2 and E3 to apply the assay to the entire ubiquitination pathway. The genetically encoded biosensor iATPSnFr1.0, also unofficially known as Bohemian, was developed for ATP fluorescence detection in vivo. We chose to tailor the “Bohemian model” towards measuring ATP hydrolysis of ubiquitination in vitro. To better understand the mechanism of fluorescence and influence of structure on activity, we have applied Bohemian to kinetic assay titrations and techniques for structural characterization including Small Angle X-ray Scattering (SAXS) and computational modeling. After optimizing Bohemian's purification, we showed Bohemian has a high affinity for ATP. We have also developed the first and only structural model of this protein. SAXS data collected on the Bohemian protein has suggested dimerization of the biosensor, which disagrees with the original intentions of the biosensor design indicated in primary literature. Future directions will look to investigate optimal assay conditions for measuring kinetics of ubiquitination and using our structural model to improve the biosensor.

Biochemical and transcript level differences between the three human phosphofructokinases show optimisation of each isoform for specific metabolic niches.

6-Phosphofructokinase-1-kinase (PFK) tetramers catalyse the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate (F16BP). Vertebrates have three PFK isoforms (PFK-M, PFK-L, and PFK-P). This study is the first to compare the kinetics, structures, and transcript levels of recombinant human PFK isoforms. Under the conditions tested PFK-M has the highest affinities for F6P and ATP (K0.5ATP 152 µM; K0.5F6P 147 µM), PFK-P the lowest affinities (K0.5ATP 276 µM; K0.5F6P 1333 µM), and PFK-L demonstrates a mixed picture of high ATP affinity and low F6P affinity (K0.5ATP 160 µM; K0.5F6P 1360 µM). PFK-M is more resistant to ATP inhibition compared with PFK-L and PFK-P (respectively, 23%, 31%, 50% decreases in specificity constants). GTP is an alternate phospho donor. Interface 2, which regulates the inactive dimer to active tetramer equilibrium, differs between isoforms, resulting in varying tetrameric stability. Under the conditions tested PFK-M is less sensitive to fructose 2,6-bisphosphate (F26BP) allosteric modulation than PFK-L or PFK-P (allosteric constants [K0.5ATP+F26BP/K0.5ATP] 1.10, 0.92, 0.54, respectively). Structural analysis of two allosteric sites reveals one may be specialised for AMP/ADP and the other for smaller/flexible regulators (citrate or phosphoenolpyruvate). Correlations between PFK-L and PFK-P transcript levels indicate that simultaneous expression may expand metabolic capacity for F16BP production whilst preserving regulatory capabilities. Analysis of cancer samples reveals intriguing parallels between PFK-P and PKM2 (pyruvate kinase M2), and simultaneous increases in PFK-P and PFKFB3 (responsible for F26BP production) transcript levels, suggesting prioritisation of metabolic flexibility in cancers. Our results describe the kinetic and transcript level differences between the three PFK isoforms, explaining how each isoform may be optimised for distinct roles.

What ATP binding does to the Ca2+ pump and how nonproductive phosphoryl transfer is prevented in the absence of Ca2+

Under physiological conditions, most Ca2+-ATPase (SERCA) molecules bind ATP before binding the Ca2+ transported. SERCA has a high affinity for ATP even in the absence of Ca2+, and ATP accelerates Ca2+ binding at pH values lower than 7, where SERCA is in the E2 state with low-affinity Ca2+-binding sites. Here we describe the crystal structure of SERCA2a, the isoform predominant in cardiac muscle, in the E2·ATP state at 3.0-Å resolution. In the crystal structure, the arrangement of the cytoplasmic domains is distinctly different from that in canonical E2. The A-domain now takes an E1 position, and the N-domain occupies exactly the same position as that in the E1·ATP·2Ca2+ state relative to the P-domain. As a result, ATP is properly delivered to the phosphorylation site. Yet phosphoryl transfer never takes place without the filling of the two transmembrane Ca2+-binding sites. The present crystal structure explains what ATP binding itself does to SERCA and how nonproductive phosphorylation is prevented in E2.

Biochemical Characterization and Allosteric Modulation by Magnesium of (Na+, K+)-ATPase Activity in the Gills of the Red Mangrove Crab Goniopsis cruentata (Brachyura, Grapsidae).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the grapsid crab Goniopsis cruentata. (Na+, K+)-ATPase activity constitutes 95% of total ATPase activity, and sucrose density centrifugation reveals an ATPase activity peak between 25 and 35% sucrose, distributed into two, partially separated protein fractions. The (Na+, K+)-ATPase α-subunit is localized throughout the ionocyte cytoplasm and has an Mr of ≈ 10kDa and hydrolyzes ATP obeying cooperative kinetics. Low (VM = 186.0 ± 9.3nmol Pi min-1 mg-1 protein and K0.5 = 0.085 ± 0.004mmol L-1) and high (VM = 153.4 ± 7.7nmol Pi min-1 mg-1 protein and K0.5 = 0.013 ± 0.0006mmol L-1) affinity ATP binding sites were characterized. At low ATP concentrations, excess Mg2+ stimulates the enzyme, triggering exposure of a high-affinity binding site that accounts for 50% of (Na+, K+)-ATPase activity. Stimulation by Mg2+ (VM = 425.9 ± 25.5nmolPimin-1mg-1 protein, K0.5 = 0.16 ± 0.01mmol L-1), K+ (VM = 485.3 ± 24.3nmol Pi min-1 mg-1 protein, K0.5 = 0.9 ± 0.05mmolL-1), Na+ (VM = 425.0 ± 23.4nmol Pi min-1mg-1 protein, K0.5 = 5.1 ± 0.3mmol L-1) and NH4+ (VM = 497.9 ± 24.9nmol Pi min-1 mg-1 protein, K0.5 = 9.7 ± 0.5mmolL-1) obeys cooperative kinetics. Ouabain inhibits up to 95% of ATPase activity with KI = 196.6 ± 9.8µmolL-1. This first kinetic characterization of the gill (Na+, K+)-ATPase in Goniopsis cruentata enables better comprehension of the biochemical underpinnings of osmoregulatory ability in this semi-terrestrial mangrove crab.

Optimizing a biosensor‐based assay for ubiquitination activation

Ubiquitination is a post‐translational modification which regulates protein degradation, DNA damage signaling, and inflammatory response. Three transfer enzymes catalyze the reaction’s pathway: E1 activating enzyme, E2 conjugating enzyme, and E3 ligating enzyme, respectively. Despite the identification of these enzymes’ structure and function, a complete understanding of their catalytic mechanisms remains unclear. Clarification of the ubiquitination mechanism could serve medicinal strategies due ubiquitin’s (Ub’s) involvement with target protein degradation. Existing assays for ubiquitination are either too imprecise, expensive, or radioactive for common usage, thus we are trying to develop a cheap, continuous assay which can make ubiquitination research more accessible. Our approach depends on the detection of ATP as E1 cleaves ATP into pyrophosphate (PPi) and AMP for Ub‐activation. Previous fluorescent assay designs for Ub‐E1 activation were attempted and examined including a terbium‐norfloxacin assay and a chloroacetaldehyde assay, but each showed limitations. The terbium‐norfloxacin assay detected ATP fluorescence, but interfered with the protein pathway, while the chloroacetaldehyde assay used organic sensors to modify ATP, but with little success. We have since turned to adapting a genetically‐encoded nucleotide biosensor, Perceval, toward measuring ATP hydrolysis of E1 in the ubiquitination pathway. Developed in the Yellen Lab at Harvard Medical School, Perceval was designed to report real‐time changes in cellular energy as a result of its competitive binding of ATP and ADP. We have worked to tailor the Perceval model towards measuring solely the ATP hydrolysis of ubiquitination in vitro, starting with E1 activation. After optimizing Perceval’s purification, we showed Perceval has a high affinity for ATP without the ADP competitive binding aspect, and ensured AMP and PPi do not interfere with the biosensor. We similarly tested solutions containing different Ub protein substrates and E1s, and found that Perceval’s affinity for ATP is not interrupted by critical components of the ubiquitination pathway. When directly applied to ubiquitination, fluorescence readings were taken at single‐time points in the ubiquitination pathway and over a continuous duration. Preliminary data suggest Perceval could be a potential biosensor for measuring Ub activation in real time. Future directions will look to further optimize assay conditions as well as test other biosensors for comparison.Support or Funding InformationJames Madison University Department of Chemistry and Biochemistry, NSF REU CHE‐1757874, Thomas and Kate Jeffress Memorial Trust, 4‐VA Organization

Saccharomyces cerevisiae polo-like kinase, Cdc5 exhibits ATP-dependent Mg2+-enhanced kinase activity in vitro

Phosphorylation of proteins on serine/threonine residues represents an important biochemical mechanism to regulate several cellular processes. Polo-like kinases (PLKs) are a family of serine-threonine kinases that play an imminent role in cell cycle regulation in yeast to humans, and thus an important therapeutic target for cancers. The present study provides insights into the enzymatic features of Saccharomyces cerevisiae PLK, Cdc5 using in vitro casein phosphorylation assays. The recombinant yeast PLK, GST-Cdc5 showed maximum casein phosphorylation activity at 30 °C, pH 9 and 45 min of incubation period. GST-Cdc5 exhibited a KM of 1.35 μM for casein, and high affinity for ATP, since addition of non-radioactive ATP chased out casein phosphorylation by radiolabeled ATP. The recombinant enzyme showed maximum kinase activity at 2.7 μM of GST-Cdc5. Casein was found to be the best in vitro substrate of GST-Cdc5 followed by BSA (Bovine Serum Albumin) and MBP (Myelin Basic Protein). Of the metal ions tested, Mg2+ (at 20 mM) was found to enhance GST-Cdc5 kinase activity, while Ca2+ (at 5 mM) and Mn2+ (at 10 mM) inhibited the same. The presence of EDTA, SDS and PMSF inhibited phosphorylation by GST-Cdc5, while DTT had no effect. The recombinant GST-Cdc5 can be used as a tool for deciphering PLKs’ structure and functions, which are still at infancy.

Positional effects of premature termination codons on the biochemical and biophysical properties of CFTR.

Biochemical and biophysical characterizations of three nonsense mutations of cystic fibrosis transmembrane conductance regulator (CFTR) associated with a severe form of cystic fibrosis (CF) reveal the importance and heterogenous effects of the position of the premature termination codon (PTC) on the CFTR protein function. Electrophysiological studies of W1282X-CFTR, whose PTC is closer to the C-terminus of CFTR, suggest the presence of both C-terminus truncated CFTR proteins that are poorly functional and read-through, full-length products. For G542X- and E60X-CFTR, the only mechanism capable of generating functional proteins is the read-through, but the outcome of read-through products is highly variable depending on the interplay between the missense mutation caused by the read-through and the structural context of the protein. Pharmacological studies of these three PTCs with various CFTR modulators suggest position-dependent therapeutic strategies for these disease-inflicting mutations. About one-third of genetic diseases and cancers are caused by the introduction of premature termination codons (PTCs). In theory, the location of the PTC in a gene determines the alternative mechanisms of translation, including premature cessation or reinitiation of translation, and read-through, resulting in differential effects on protein integrity. In this study, we used CFTR as a model system to investigate the positional effect of the PTC because of its well-understood structure-function relationship and pathophysiology. The characterization of three PTC mutations, E60X-, G542X- and W1282X-CFTR revealed heterogenous effects of these PTCs on CFTR function. The W1282X mutation results in both C-terminus truncated and read-through proteins that are partially or fully functional. In contrast, only the read-through protein is functional with E60X- and G542X-CFTR, although abundant N-terminus truncated proteins due to reinitiation of translation were detected in E60X-CFTR. Single-channel studies of the read-through proteins of E60X- and G542X-CFTR demonstrated that both mutations have a single-channel amplitude similar to wild type (WT), and good responses to high-affinity ATP analogues, suggesting intact ion permeation pathways and nucleotide binding domains (NBDs), albeit with reduced open probability (Po ). The comparison of the Po of these mutations with the proposed missense mutations revealed potential identities of the read-through products. Importantly, a majority of the functional protein studied responds to CFTR modulators like GLPG1837 and Lumacaftor. These results not only expand current understanding of the molecular (patho)physiology of CFTR, but also infer therapeutic strategies for different PTC mutations at large.

A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin.

Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP–PNP–DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP–PNP–DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.

Characterization of Mitochondrial YME1L Protease Oxidative Stress-Induced Conformational State

Oxidative stress is a common challenge to mitochondrial function where reactive oxygen species are capable of significant organelle damage. The generation of mitochondrial reactive oxygen species occurs in the inner membrane and matrix compartments as a consequence of subunit function in the electron transport chain and citric acid cycle, respectively. Maintenance of mitochondrial proteostasis and stress response is facilitated by compartmentalized proteases that couple the energy of ATP hydrolysis to unfolding and the regulated removal of damaged, misfolded, or aggregated proteins. The mitochondrial protease YME1L functions in the maintenance of proteostasis in the intermembrane space. YME1L is an inner membrane-anchored hexameric protease with distinct N-terminal, transmembrane, AAA+ (ATPases associated with various cellular activities), and C-terminal M41 zinc-dependent protease domains. The effect of oxidative stress on enzymes such as YME1L tasked with maintaining proteostasis is currently unclear. We report here that recombinant YME1L undergoes a reversible conformational change in response to oxidative stress that involves the interaction of one hydrogen peroxide molecule per YME1L monomer with affinities equal to 31 ± 2 and 26 ± 1 mM for conditions lacking or including nucleotide, respectively. Our data also reveal that oxidative stress does not significantly impact nucleotide binding equilibria, but does stimulate a 2-fold increase in the rate constant for high-affinity ATP binding from (8.9 ± 0.2) × 105 M−1 s−1 to (1.5 ± 0.1) × 106 M−1 s−1. Taken together, these data may suggest a mechanism for the regulated processing of YME1L by other inner membrane proteases such as OMA1.

ATP boosts lit state formation and activity of Arabidopsis cryptochrome 2.

Cryptochrome (cry) blue light photoreceptors have important roles in the regulation of plant development. Their photocycle includes redox changes of their flavin adenine dinucleotide (FAD) chromophore, which is fully oxidised in the dark state and semi-reduced in the signalling-active lit state. The two Arabidopsis thaliana cryptochromes, cry1 and cry2, and the plant-type cryptochrome CPH1 from Chlamydomonas rheinhardtii bind ATP and other nucleotides. Binding of ATP affects the photocycle of these photoreceptors and causes structural alterations. However, the exact regions that undergo structural changes have not been defined, and most importantly it is not known whether ATP binding affects the biological activity of these photoreceptors in planta. Here we present studies on the effect of ATP on Arabidopsis cry2. Recombinant cry2 protein showed a high affinity for ATP (KD of 1.09±0.48μm). Binding of ATP and other adenines promoted photoreduction of the FAD chromophore invitro and caused structural changes, particularly in α-helix 21 which links the photosensory domain with the C-terminal extension. The constructed cry2Y399A mutant was unable to bind ATP and did not show enhancement of photoreduction by ATP. When this mutant gene was expressed in Arabidopsis null cry2 mutant plants it retained some biological activity, which was, however, lower than that of the wild type. Our results indicate that binding of ATP to cry2, and most likely to other plant-type cryptochromes, is not essential but boosts the formation of the signalling state and biological activity.

Anti-diabetic drug binding site in a mammalian KATP channel revealed by Cryo-EM.

Sulfonylureas are anti-diabetic medications that act by inhibiting pancreatic KATP channels composed of SUR1 and Kir6.2. The mechanism by which these drugs interact with and inhibit the channel has been extensively investigated, yet it remains unclear where the drug binding pocket resides. Here, we present a cryo-EM structure of a hamster SUR1/rat Kir6.2 channel bound to a high-affinity sulfonylurea drug glibenclamide and ATP at 3.63 Å resolution, which reveals unprecedented details of the ATP and glibenclamide binding sites. Importantly, the structure shows for the first time that glibenclamide is lodged in the transmembrane bundle of the SUR1-ABC core connected to the first nucleotide binding domain near the inner leaflet of the lipid bilayer. Mutation of residues predicted to interact with glibenclamide in our model led to reduced sensitivity to glibenclamide. Our structure provides novel mechanistic insights of how sulfonylureas and ATP interact with the KATP channel complex to inhibit channel activity.

The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase.

The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the ε subunit from bacterial ATP synthases.

Transient Kinetic Analysis of ATP Hydrolysis by the CCT/TRiC Chaperonin

The chaperonin-containing t-complex polypeptide 1 (CCT, also known as TRiC) assists protein folding in an ATP-dependent manner. CCT/TRiC was mixed rapidly with different concentrations of ATP, and the amount of phosphate formed upon ATP hydrolysis was measured as a function of time using the coumarin-labeled phosphate-binding protein method. Two burst phases were observed, followed by a lag phase and then a linear steady-state phase of ATP hydrolysis. The phases were assigned by (i) determining their dependence on ATP and K+ concentrations and (ii) by measuring their sensitivity to the mutation Gly345→Asp in subunit CCT4, which decreases cooperativity in ATP binding. The values of the observed rate constants corresponding to the burst phases are found to decrease with increasing ATP and K+ concentrations, thereby indicating that the apo state of CCT/TRiC is in equilibrium between several conformations and that “conformational selection” by ATP takes place before hydrolysis. The amplitude of the lag phase, which follows, decreases with increasing ATP concentrations, thus indicating that it reflects a transition between states with low affinity for ATP and a state with high affinity for ATP that is predominant under steady-state conditions. A kinetic model based on the data is suggested, in which CCT/TRiC is in equilibrium between a relatively large number of states that are distinguished kinetically, in agreement with its proposed sequential allosteric mechanism.

Biochemical and biophysical studies of Trypanosoma cruzi phosphofructokinase as a target against Chagas disease

BackgroundTrypanosoma cruzi is a protozoan parasite that causes Chagas disease, a chronic infection affecting the sympathetic nervous system; no effective non-toxic treatments are available. Inhibitors of trypanosomatid glycolytic enzymes are promising therapeutic targets, as shown by research on T brucei phosphofructokinase (PFK). The aim of this study was to investigate the structure, function, and regulation of T cruzi PFK, to provide insights into the suitability of this enzyme as a drug target. MethodsBL21(DE3) Escherichia coli cells were transformed with pET28a plasmid containing cDNA for T cruzi PFK with N-terminal hexahistidine tag, and induced with isopropyl-β-D-thiogalactopyranoside. Pellets were lysed by liquid homogenisation and purified through immobilised metal ion affinity chromatography and size exclusion chromatography. Kinetic values and inhibition were determined with an auxiliary enzyme-linked assay with pyruvate kinase and lactate dehydrogenase. Crystallisation was done via the hanging drop method in 24-well plates at 18°C. FindingsT cruzi PFK was fully purified and verified with mass spectrometry (Mascot score of 241). Dynamic light scattering indicated a homogenous sample of molecular weight 239 kDa. Apo-enzyme and holo-enzyme crystals were produced (cofactors ATP or AMP). Maximum specific activity (Vmax) was 58·9 μmol/min per mg. ATP binding was hyperbolic (Michaelis-Menten kinetics) with KmATP 52·9 μM. F6P binding was sigmoidal (allosteric sigmoidal kinetics) with KhalfF6P 429 μM. GTP was an alternative substrate but showed reduced activity and affinity compared with ATP. AMP was an allosteric activator, reducing KmATP to 15 μM. F16BP was inhibitory above 2·5 mM, but no other metabolites altered activity. Novel small molecule inhibitors demonstrated sub-50 nanomolar IC50 values for T cruzi PFK, and were inactive against human PFK. InterpretationThe biophysical results indicate that T cruzi PFK is tetrameric, similar to other PFKs. Compared with T brucei PFK, T cruzi PFK has a higher affinity for ATP and F6P and similar Vmax. The relative lack of allosteric regulators can be explained by the localisation of T cruzi PFK within glycosomes. The physiological significance of GTP use is doubtful given the lack of a glycosomal mechanism for regenerating GTP from GDP. Novel inhibitors bound allosterically, showing that they have great promise as candidate drugs. FundingWellcome Trust.