Probing the ATP hydrolysis cycle of the ABC multidrug transporter LmrA by pulsed EPR spectroscopy

Members of the ATP binding cassette (ABC) transporter superfamily translocate various types of molecules across the membrane at the expense of ATP. This requires cycling through a number of catalytic states. Here, we report conformational changes throughout the catalytic cycle of LmrA, a homodimeric multidrug ABC transporter from L. lactis. Using site-directed spin labeling and pulsed electron-electron double resonance (PELDOR/DEER) spectroscopy, we have probed the reorientation of the nucleotide binding domains and transmembrane helix 6 which is of particular relevance to drug binding and part of the dimerization interface. Our data show that LmrA samples a very large conformational space in its apo state, which is significantly reduced upon nucleotide binding. ATP binding but not hydrolysis is required to trigger this conformational change, which results in a relatively fixed orientation of both the nucleotide binding domains and transmembrane helices 6. This orientation is maintained throughout the ATP hydrolysis cycle until the protein cycles back to its apo state. Our data present strong evidence that switching between two dynamically and structurally distinct states is required for substrate translocation.     

Time-Resolved Mn2+−NO and NO−NO Distance Measurements Reveal That Catalytic Asymmetry Regulates Alternating Access in an ABC Transporter**

ATP-binding cassette (ABC) transporters shuttle diverse substrates across biological membranes. Transport is often achieved through a transition between an inward-facing (IF) and an outward-facing (OF) conformation of the transmembrane domains (TMDs). Asymmetric nucleotide-binding sites (NBSs) are present among several ABC subfamilies and their functional role remains elusive. Here we addressed this question using concomitant NO−NO, Mn²⁺−NO, and Mn²⁺−Mn²⁺ pulsed electron–electron double-resonance spectroscopy of TmrAB in a time-resolved manner. This type-IV ABC transporter undergoes a reversible transition in the presence of ATP with a significantly faster forward transition. The impaired degenerate NBS stably binds Mn²⁺−ATP, and Mn²⁺ is preferentially released at the active consensus NBS. ATP hydrolysis at the consensus NBS considerably accelerates the reverse transition. Both NBSs fully open during each conformational cycle and the degenerate NBS may regulate the kinetics of this process.     

Computational Insights into Allosteric Conformational Modulation of P-Glycoprotein by Substrate and Inhibitor Binding

The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) is a physiologically essential membrane protein that protects many tissues against xenobiotic molecules, but limits the access of chemotherapeutics into tumor cells, thus contributing to multidrug resistance. The atomic-level mechanism of how substrates and inhibitors differentially affect the ATP hydrolysis by P-gp remains to be elucidated. In this work, atomistic molecular dynamics simulations in an explicit membrane/water environment were performed to explore the effects of substrate and inhibitor binding on the conformational dynamics of P-gp. Distinct differences in conformational changes that mainly occurred in the nucleotide-binding domains (NBDs) were observed from the substrate- and inhibitor-bound simulations. The binding of rhodamine-123 can increase the probability of the formation of an intermediate conformation, in which the NBDs were closer and better aligned, suggesting that substrate binding may prime the transporter for ATP hydrolysis. By contrast, the inhibitor QZ-Leu stabilized NBDs in a much more separated and misaligned conformation, which may result in the deficiency of ATP hydrolysis. The significant differences in conformational modulation of P-gp by substrate and inhibitor binding provided a molecular explanation of how these small molecules exert opposite effects on the ATPase activity. A further structural analysis suggested that the allosteric communication between transmembrane domains (TMDs) and NBDs was primarily mediated by two intracellular coupling helices. Our computational simulations provide not only valuable insights into the transport mechanism of P-gp substrates, but also for the molecular design of P-gp inhibitors.     

ENERGY-REGULATED FUNCTIONAL TRANSITIONS OF CHLOROPLAST ATPASE

The functional transitions of the membrane-bound chloroplast ATPase (CF₁) as influenced by low ADP and uncoupler concentrations are investigated by measurements of initial and steady-state ATP hyrolysis and concomitant membrane energization. Following activation of latent ATP hydrolysis by light in the presence of dithioerythritol, the resulting steady-state ATP hydrolysis depends on the dark-period ( t _d) bteween light activation and ATP addition. ADP, added during t _d, inhibits this activity ( K _i about 2 μ M ) and induces a lag in the onset of ATP hydrolysis. The extent of membrane energization as monitored by an aminoacridine fluorescent probe is proportional to the ATPase activity.
An uncoupler amplifies the inhibitory effect of ADP if added during f _d, whereas it induces the normal stimulation of ATP hydrolysis in the absence of ADP. The ADP effect, which is different from product inhibition, is interpreted as a conformational interaction with CF₁ causing an increase of the energy threshold required for the inactive → active transition of the CF₁ molecules. These results are in harmony with currently proposed models of CF₁ regulation by adenine nucleotides based on binding studies.
The inactive → active transition of CF₁ conformation is investigated by analysis of the lag in the onset of ATP hydrolysis at different ADP concentrations and by means of varied light pulses and single-turnover flashes, using the electric potential indicating absorption change at 515 nm as a probe for the onset of ATP hydrolysis. The half-time of the process leading to fully (re)activated ATP hydrolysis is about 0.25 s. The ATP-dependent flash-induced inactive → active transition occurs within a few turnovers of electron flow.     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate.

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, KD = 1-2 muM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (KI = 240-300 muM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This "tightly bound" ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetics studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, Pi can maintain the active form of the enzyme.     

Understanding cAMP-dependent allostery by NMR spectroscopy: comparative analysis of the EPAC1 cAMP-binding domain in its apo and cAMP-bound states

cAMP (adenosine 3',5'-cyclic monophosphate) is a ubiquitous second messenger that activates a multitude of essential cellular responses. Two key receptors for cAMP in eukaryotes are protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC), which is a recently discovered guanine nucleotide exchange factor (GEF) for the small GTPases Rap1 and Rap2. Previous attempts to investigate the mechanism of allosteric activation of eukaryotic cAMP-binding domains (CBDs) at atomic or residue resolution have been hampered by the instability of the apo form, which requires the use of mixed apo/holo systems, that have provided only a partial picture of the CBD apo state and of the allosteric networks controlled by cAMP. Here, we show that, unlike other eukaryotic CBDs, both apo and cAMP-bound states of the EPAC1 CBD are stable under our experimental conditions, providing a unique opportunity to define at an unprecedented level of detail the allosteric interactions linking two critical functional sites of this CBD. These are the phosphate binding cassette (PBC), where cAMP binds, and the N-terminal helical bundle (NTHB), which is the site of the inhibitory interactions between the regulatory and catalytic regions of EPAC. Specifically, the combined analysis of the cAMP-dependent changes in chemical shifts, 2 degrees structure probabilities, hydrogen/hydrogen exchange (H/H) and hydrogen/deuterium exchange (H/D) protection factors reveals that the long-range communication between the PBC and the NTHB is implemented by two distinct intramolecular cAMP-signaling pathways, respectively, mediated by the beta2-beta3 loop and the alpha6 helix. Docking of cAMP into the PBC perturbs the NTHB inner core packing and the helical probabilities of selected NTHB residues. The proposed model is consistent with the allosteric role previously hypothesized for L273 and F300 based on site-directed mutagenesis; however, our data show that such a contact is part of a significantly more extended allosteric network that, unlike PKA, involves a tight coupling between the alpha- and beta-subdomains of the EPAC CBD. The proposed mechanism of allosteric activation will serve as a basis to understand agonism and antagonism in the EPAC system and provides also a general paradigm for how small ligands control protein-protein interfaces.     

Molecular phylogenetic study and expression analysis of ATP-binding cassette transporter gene family in Oryza sativa in response to salt stress

ATP-binding cassette (ABC) transporter is a large gene superfamily that utilizes the energy released from ATP hydrolysis for transporting myriad of substrates across the biological membranes. Although many investigations have been done on the structural and functional analysis of the ABC transporters in Oryza sativa, much less is known about molecular phylogenetic and global expression pattern of the complete ABC family in rice. In this study, we have carried out a comprehensive phylogenetic analysis constructing neighbor-joining and maximum-likelihood trees based on various statistical methods of different ABC protein subfamily of five plant lineages including Chlamydomonas reinhardtii (green algae), Physcomitrella patens (moss), Selaginella moellendorffii (lycophyte), Arabidopsis thaliana (dicot) and O. sativa (monocot) to explore the origin and evolutionary patterns of these ABC genes. We have identified several conserved motifs in nucleotide binding domain (NBD) of ABC proteins among all plant lineages during evolution. Amongst the different ABC protein subfamilies, ‘ABCE’ has not yet been identified in lower plant genomes (algae, moss and lycophytes). The result indicated that gene duplication and diversification process acted upon these genes as a major operative force creating new groups and subgroups and functional divergence during evolution. We have demonstrated that rice ABCI subfamily consists of only half size transporters that represented highly dynamic members showing maximum sequence variations among the other rice ABC subfamilies. The evolutionary and the expression analysis contribute to a deep insight into the evolution and diversity of rice ABC proteins and their roles in response to salt stress that facilitate our further understanding on rice ABC transporters.     

Testing for violations of microscopic reversibility in ATP-sensitive potassium channel gating

In pancreatic beta cells, insulin secretion is tightly controlled by the cells' metabolic state via the ATP-sensitive potassium (KATP) channel. ATP is a key mediator in this signaling process, where its role as an inhibitor of KATP channels has been extensively studied. Since the channel contains an ATPase as an accessory subunit, the possibility that ATP hydrolysis mediates KATP channel opening has also been proposed. However, a rigorous test of coupling between ATP hydrolysis and channel gating has not previously been performed. In the present work, we examine whether KATP channel gating obeys detailed balance in order to determine whether ATP hydrolysis is strongly coupled to the gating of the KATP channel. Single-channel records were obtained from inside-out patches of transiently transfected HEK-293 cells. Channel activity in membrane patches with exactly one channel shows no violations of microscopic reversibility. Although KATP channel gating shows long closed times on the time scale where ATP hydrolysis takes place, the time symmetry of channel gating indicates that it is not tightly coupled to ATP hydrolysis. This lack of coupling suggests that channel gating operates close to equilibrium; although detailed balance is not expected to hold for ATP hydrolysis, it still does so in channel gating. On the basis of these results, the function of the ATPase active site in channel gating may be to sense nucleotides by differential binding of ATP and ADP, rather than to drive a thermodynamically unfavorable conformational change.     

Hierarchical dynamics in allostery following ATP hydrolysis monitored by single molecule FRET measurements and MD simulations

We report on a study that combines advanced fluorescence methods with molecular dynamics (MD) simulations to cover timescales from nanoseconds to milliseconds for a large protein. This allows us to delineate how ATP hydrolysis in a protein causes allosteric changes at a distant protein binding site, using the chaperone Hsp90 as test system. The allosteric process occurs via hierarchical dynamics involving timescales from nano- to milliseconds and length scales from Ångstroms to several nanometers. We find that hydrolysis of one ATP is coupled to a conformational change of Arg380, which in turn passes structural information via the large M-domain α-helix to the whole protein. The resulting structural asymmetry in Hsp90 leads to the collapse of a central folding substrate binding site, causing the formation of a novel collapsed state (closed state B) that we characterise structurally. We presume that similar hierarchical mechanisms are fundamental for information transfer induced by ATP hydrolysis through many other proteins.

We report on a study that combines advanced fluorescence methods with molecular dynamics simulations to cover timescales from nanoseconds to milliseconds for a large protein, the chaperone Hsp90.     

Density functional calculations of ATP systems. 2. ATP hydrolysis at the active site of actin

The hydrolysis of adenosine 5'-triphosphate (ATP) at the active site of actin has been studied using density functional calculations. The active site is modeled by the triphosphate tail of ATP, an Mg cation, surrounding water molecules, and the nearby protein residues. Four reaction paths have been followed by constraining coordinates that allow phosphate stretching, nucleophilic attack of the catalytic water, and OH(-) formation via water deprotonation. The lowest-energy barrier (21.0 kcal/mol) is obtained for a dissociative reaction where the terminal phosphate breaks on approaching the catalytic water, followed by proton release via a proton wire mechanism. A higher barrier (39.6 kcal/mol) results for an associative reaction path where OH(-) is formed first, with a pentacoordinated phosphorus atom (P-O distances 2.1 A). Stretching the terminal bridging P-O bond results in bond rupture at 2.8 A with an energy barrier of 28.8 kcal/mol. The residues Gln137 and His161 are not important in the reactions, but insight into their roles in vivo has been obtained. The favored coordination of the end products H(2)PO(4)(-) and ADP(3-) includes a hydrogen bond and an O-Mg-O bridge between the phosphates as well as a hydrogen bond between H(2)PO(4)(-) and the Ser14 side chain. The total energy is 2.1 kcal/mol lower than in the initial reactants. Classical simulations of ATP- and ADP.P(i)-actin show few hydrolysis-induced differences in the protein structure, indicating that phosphate migration is necessary for a change in conformation.     

Rational design of allosteric ribozymes

Background: Efficient operation of cellular processes relies on the strict control that each cell exerts over its metabolic pathways. Some protein enzymes are subject to allosteric regulation, in which binding sites located apart from the enzyme's active site can specifically recognize effector molecules and alter the catalytic rate of the enzyme via conformational changes. Although RNA also performs chemical reactions, no ribozymes are known to operate as true allosteric enzymes in biological systems. It has recently been established that small-molecule receptors can readily be made of RNA, as demonstrated by the in vitro selection of various RNA aptamers that can specifically bind corresponding ligand molecules. We set out to examine whether the catalytic activity of an existing ribozyme could be brought under the control of an effector molecule by designing conjoined aptamer-ribozyme complexes.Results: By joining an ATP-binding RNA to a self-cleaving ribozyme, we have created the first example of an allosteric ribozyme that has a catalytic rate that can be controlled by ATP. A 180-fold reduction in rate is observed upon addition of either adenosine or ATP, but no inhibition is detected in the presence of dATP or other nucleoside triphosphates. Mutations in the aptamer domain that are expected to eliminate ATP binding or that increase the distance between aptamer and ribozyme domains result in a loss of ATP-specific allosteric control. Using a similar design approach, allosteric hammerhead ribozymes that are activated in the presence of ATP were created and another ribozyme that can be controlled by theophylline was created.Conclusions: The catalytic features of these conjoined aptamer-ribozyme constructs demonstrate that catalytic RNAs can also be subject to allosteric regulation — a key feature of certain protein enzymes. Moreover, by using simple rational design strategies, it is now possible to engineer new catalytic polynucleotides which have rates that can be tightly and specifically controlled by small effector molecules.     

Probing conformational variations at the ATPase site of the RNA helicase DbpA by high-field electron-nuclear double resonance spectroscopy

The RNA helicase DbpA promotes RNA remodeling coupled to ATP hydrolysis. It is unique because of its specificity to hairpin 92 of 23S rRNA (HP92). Although DbpA kinetic pathways leading to ATP hydrolysis and RNA unwinding have been recently elucidated, the molecular (atomic) basis for the coupling of ATP hydrolysis to RNA remodeling remains unclear. This is, in part, due to the lack of detailed structural information on the ATPase site in the presence and absence of RNA in solution. We used high-field pulse ENDOR (electron-nuclear double resonance) spectroscopy to detect and analyze fine conformational changes in the protein's ATPase site in solution. Specifically, we substituted the essential Mg(2+) cofactor in the ATPase active site for paramagnetic Mn(2+) and determined its close environment with different nucleotides (ADP, ATP, and the ATP analogues ATPγS and AMPPnP) in complex with single- and double-stranded RNA. We monitored the Mn(2+) interactions with the nucleotide phosphates through the (31)P hyperfine couplings and the coordination by protein residues through (13)C hyperfine coupling from (13)C-enriched DbpA. We observed that the nucleotide binding site of DbpA adopts different conformational states upon binding of different nucleotides. The ENDOR spectra revealed a clear distinction between hydrolyzable and nonhydrolyzable nucleotides prior to RNA binding. Furthermore, both the (13)C and the (31)P ENDOR spectra were found to be highly sensitive to changes in the local environment of the Mn(2+) ion induced by the hydrolysis. More specifically, ATPγS was efficiently hydrolyzed upon binding of RNA, similar to ATP. Importantly, the Mn(2+) cofactor remains bound to a single protein side chain and to one or two nucleotide phosphates in all complexes, whereas the remaining metal coordination positions are occupied by water. The conformational changes in the protein's ATPase active site associated with the different DbpA states occur in remote coordination shells of the Mn(2+) ion. Finally, a competitive Mn(2+) binding site was found for single-stranded RNA construct.     

Mathematical modeling of kinetics of adenosine-5’-triphosphate hydrolysis catalyzed by the Zn2+ ion in the pH range 8.5–9.0

The kinetics of adenosine-5’-triphosphate (ATP) hydrolysis catalyzed by Zn²⁺ at pH 8.5–9.0 is analyzed by numerical simulation. The rates of product formation (adenosine diphosphate (ADP) and adenosine monophosphate (AMP)) are determined by a conformational transformation. In the sequence of steps of mutual transformations of cyclic (Cy) pH-dependent species, which are active in ATP hydrolysis to ADP, and open (Op) species, the rate-limiting step is the slow isomerization of ZnATP²-complexes. This slow step is determined by the abstraction of the OH^- group from a pentacovalent intermediate catalyzed by H₃O⁺. In the Op species,Zn ²⁺ is bound to the phosphate chain. In the Cy species, which can be hydrolized to ADP, Zn²⁺ coordinates a nitrogen atom in position 7 and γ-phosphate. The mutual transformations of conformers occur via pentacovalent intermediates with the participation of γ-phosphorus and include pseudotransformations. In the direct transformation CyOH^-⦚r OpOH^-, pseudotransformation is a rate-controlling step. The deprotonated open monomeric form OpOH^- is inactive in hydrolysis. Within the framework of the dimeric model and a more complex model that accounts for the role of trimeric associates ZnATP^2-, the general scheme of intermediate transformations is considered that accounts for the existence of a pH-independent pathway of hydrolysis. The rate and equilibrium constants are estimated. Concentration profiles for intermediate products during hydrolysis are described.     

Exploring sites on mitochondrial ATPase for catalysis, regulation, and inhibition.

Evidence is presented that mitochondrial ATPase has two types of sites that bind adenine nucleotides. The catalytic site, C, binds the substrates ATP, GTP, or ITP and the inhibitor guanylyl imidodiphosphate (GMP-PNP). A second type of site, R, binds ATP, ADP, adenylyl imidodiphosphate (AMP-PNP), and the chromium complexes of ATP or ADP. All of these substances binding to the R site inhibit the hydrolysis of ATP in a competitive manner; their inhibition of hydrolysis of ITP and GTP is noncompetitive. GMP-PNP inhibits oxidative phosphorylation in submitochondrial particles but AMP-PNP does not. The localization on mitochondrial membranes of sites for the binding of various antibiotics that inhibit oxidative phosphorylation is discussed.     

布朗动力学理论模拟动态肌动蛋白纤维的增长

肌动蛋白的聚合耦合三磷酸腺酐(ATP)分子水解成二磷酸腺苷(ADP)分子和磷酸(Pi)的释放两个过程. 因此, 肌动蛋白纤维上的原聚体存在三种不同状态, 即分别结合ATP, ADP/Pi和ADP分子. 原聚体的不同状态导致纤维具有不同的空间图谱, 这些状态的空间分布将影响纤维的各种行为. 为此,建立了相应的分子模型,在布朗动力学模拟中实现了遵循时间演化的连续马尔可夫随机过程的解聚和水解反应; 重点阐述了如何实现纤维两端的聚合和解聚达到化学平衡的方法, 并系统研究了纤维在结合ATP分子的肌动蛋白单体溶液中的增长行为.     

Intrinsic Apyrase-Like Activity of Cerium-Based Metal–Organic Frameworks (MOFs): Dephosphorylation of Adenosine Tri- and Diphosphate

Apyrase is an important family of extracellular enzymes that catalyse the hydrolysis of high-energy phosphate bonds (HEPBs) in ATP and ADP, thereby modulating many physiological processes and driving life activities. Herein, we report an unexpected discovery that cerium-based metal–organic frameworks (Ce-MOFs) of UiO-66(Ce) have intrinsic apyrase-like activity for ATP/ADP-related physiological processes. The abundant Ce^III/Ce^IV couple sites of Ce-MOFs endow them with the ability to selectively catalyse the hydrolysis of HEPBs of ATP and ADP under physiological conditions. Compared to natural enzymes, they could resist extreme pH and temperature, and present a broad range of working conditions. Based on this finding, a significant inhibitory effect on ADP-induced platelet aggregation was observed upon exposing the platelet-rich plasma (PRP) to the biomimetic UiO-66(Ce) films, prefiguring their wide application potentials in medicine and biotechnology.     

Myosin-catalyzed ATP hydrolysis elucidated by 31P NMR kinetic studies and 1H PFG-diffusion measurements

We conducted ³¹P NMR kinetic studies and ¹H-diffusion measurements on myosin-catalyzed hydrolysis of adenosine triphosphate (ATP) under varied conditions. The data elucidate well the overall hydrolysis rate and various factors that significantly impact the reaction. We found that the enzymatic hydrolysis of ATP to adenosine diphosphate (ADP) was followed by ADP hydrolysis, and different nucleotides such as ADP and guanosine triphosphate acted as competitors of ATP. Increasing ATP or Mg²⁺ concentration resulted in decreased hydrolysis rate, and such effect can be related to the decrease of ATP diffusion constants. Below 50 °C, the hydrolysis was accelerated by increasing temperature following the Arrhenius’ law, but the hydrolysis rate was significantly lowered at higher temperature (~60 °C), due to the thermal–denaturation of myosin. The optimal pH range was around pH 6–8. These results are important for characterization of myosin-catalyzed ATP hydrolysis, and the method is also applicable to other enzymatic nucleotide reactions.     

腺苷酸激酶催化循环后期Mg2+转移的分子动力学模拟

腺苷酸激酶是一个包含三个结构域(LID结构域、NMP结构域和CORE结构域)的蛋白质分子,其主要作用是催化化学反应Mg²⁺ + ATP + AMP ⇌ 2ADP + Mg²⁺,进而将细胞内ATP分子的浓度维持在合适的范围内。在腺苷酸激酶催化上述化学反应的过程中,需要有Mg²⁺的参与。最近的实验发现Mg²⁺不仅参与上述反应的化学步骤,而且对化学反应发生后底物的释放过程至关重要。已有晶体结构数据显示,在催化循环过程的化学反应步骤完成后,一个Mg²⁺可同时和分别位于LID结构域及NMP结构域的两个ADP分子配位。然而,在底物的释放与分离过程中, Mg²⁺可能只与其中一个ADP分子结合。由于Mg²⁺与ADP分子的结合情况会在很大程度上影响作为催化循环限速步骤的底物释放过程,因此人们有必要研究清楚在底物释放前Mg²⁺与催化产物ADP分子的配位情况,即Mg²⁺更倾向于与LID结构域的ADP分子结合还是与NMP结构域的ADP分子结合。本文中,我们对催化反应后底物释放前的酶-底物复合物(包含酶、两个ADP分子以及Mg²⁺)做了分子动力学模拟研究。我们基于metadynamics方法得到了Mg²⁺在两个ADP分子间转移的自由能面,发现在底物分离与释放过程中, Mg²⁺更倾向于与LID结构域的ADP分子结合。只有当LID结构域的ADP分子被质子化,同时NMP结构域的ADP分子处于去质子化状态时, Mg²⁺才会倾向于与NMP结构域的ADP分子结合。另外,我们也刻画了Mg²⁺转移过程中配体交换与脱水过程。本工作的研究结果有助于理解腺苷酸激酶催化循环后期的分子过程。     

Cellular uptake of molybdenum and tungsten

Cells acquire molybdenum and tungsten as their highly soluble oxoanions, Mo^VIO₄^2? or W^VIO₄^2?, which they internalize by means of an active (i.e. energy requiring) transmembrane importer, for subsequent conversion into the metalloenzyme cofactors Moco or Wco (and FeMoco in nitrogen fixers). This import system has been studied as one of the models for the functioning of the protein complex superfamily of ABC (ATP binding cassette) transporters, but its mechanistic details are presently not clear. The complex exhibits interesting variants, known as the microbial Mod, Tup, and Wtp system, and the – less well defined – eukaryotic MOT1 system, which mutually differ in oxoanion coordination chemistry and in the control of intracellular Mo/W levels. This evolutionary diversity of Mo/W transporters has resulted in confusing nomenclature whose rectification is here proposed.     

A mass spectrometry based functional assay for the quantitative assessment of ABC transporter activity

ATP‐Binding Cassette (ABC) transporters are highly expressed in pharmacological barriers limiting the access of drugs to their targets. Since characterization of a compound as a transporter substrate or inhibitor bears significant consequences in drug development, there is a great need for reliable tools that enable the rapid analysis of the transport susceptibility of drugs. Here we describe a simple but very efficient high‐performance liquid chromatography/mass spectrometry (HPLC/MS) assay for measuring the ABC transporter‐dependent vesicular transport of compounds. In addition, we provide evidence that the requirement for sample preparation can be minimized using desorption electrospray ionization (DESI)‐MS, paving the way for a direct, high‐throughput investigation of drug‐transporter interactions. Copyright © 2009 John Wiley & Sons, Ltd.