ATP hydrolysis

ATP-ADP exchange was estimated i n the presence of plasma membrane H+-ATPase of oat (Avena sativa) roots partially purified with Triton X-1 O0 by measuring ['4C]ATP formation from [14CIADP. Most studies were done at 0°C. At p H 6.0 the exchange showed: (a) Mg2+ requirement with a biphasic response giving maximal activity at 152 p~ and (b) insensitivity t o ionic strength, [Na+], and [K+l. ATP and ADP dependence were analyzed with a model in which nucleotide-enzyme interactions are at rapid-random equilibrium, whereas E,ATP E,P-ADP transitions occur in steady state. The results indicated competition between ADP and ATP for the catalytic site, whereas ATP interaction with the ADP site was extremely weak. At 0°C the exchange showed a 3-fold p H increase, from p H 5.5 t o 9.0. At an alkaline p H the reaction was not affected by sodium azide and carbonyl cyanide p-trifluometoxyphenylhydrazone, had a biphasic response t o Mgz+ (maximal at 51 3 pm), and was insensitive to ionic strength. At 20°C ATP-ADP exchange was p H insensitive. At both temperatures ATP hydrolysis displayed a bell-shaped response, with a maximum around p H 6.0 t o 6.5. Because no adenylate kinase activity was detected under any condition, these results demonstrate the existence of an ATP-ADP exchange reaction catalyzed by the plant H+-ATPase. The PM H+-ATPase from higher plants belongs to the family of (E-P)ATPases that catalyze cation transport across membranes . These enzymes are also involved in Na+, K+, and Ca2+ transport (Goffeau and Slayman, 1981; and share a basic part of the reaction cycle involving the formation and subsequent hydrolysis of an acid-stable acyl-phosphate intermediate, E-P. The cycles also include the shifting between two conformational states: (a) E,, which represents the enzyme with a high affinity for the substrate and the cytoplasmic cation to be transported; and (b) E,, which has a low affinity for the substrate and the intracellular-transported cations. In coupled systems such as the Na+,K+-ATPase system, the E, state has a high affinity for the extracellular-transported cation (Glynn, 1985). The formation of E-P from ATP leads to the release of ADP and, later, Pi in a uni-bi-ordered reaction. In a reversible cycle, as is the case with a11 studied This work was supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas, Consejo de Investigaciones Científicas y Technológicas de la Provincia de Córdoba (3511/95), and Volkswagen-Stiftung (I/72 122). This work is part of the fulfillment for G.H. for a doctoral degree.

PglK is an ABC transporter that flips a lipid-linked oligosaccharide (LLO) that serves as a donor in protein N-glycosylation. Previous structures revealed two inward-facing conformations, both with very large separations of the nucleotide binding domains (NBDs), and a closed, ADP-bound state that featured an occluded cavity. To investigate additional states, we developed conformation-sensitive, single-domain camelid nanobodies (Nb) and studied their effect on PglK activity. Biochemical, structural, and mass spectrometric analyses revealed that one inhibitory Nb binds as a single copy to homodimeric PglK. The co-crystal structure of this Nb and ADP-bound PglK revealed a new, narrowly inward-open conformation. Rather than inducing asymmetry in the PglK homodimer, the binding of one Nb results in steric constraints that prevent a second Nb to access the symmetry-related site in PglK. The Nb performed its inhibitory role by a “sticky-doorstop” mechanism, where inhibition of ATP hydrolys...

In order to quantify the intrinsic dynamics associated with the tip of a GTP-cap under semi-confined conditions, such as those within a neuronal cone and at a kinetochoremicrotubule interface, we propose a novel quantitative concept of critical nano local GTPtubulin concentration (CNLC). A simulation of a rate constant of GTP-tubulin hydrolysis, under varying conditions based on this concept, generates results in the range of 0-420 s -1 . These results are in agreement with published experimental data, validating our model. The major outcome of this model is the prediction of 11 random and distinct outbursts of GTP hydrolysis per single layer of a GTP-cap. GTP hydrolysis is accompanied by an energy release and the formation of discrete expanding zones, built by less-stable, skewed GDP-tubulin subunits. We suggest that the front of these expanding zones within the walls

Fragmented flagellar axonemes of sand dollar spermatozoa were reactivated by rapid photolysis of caged ATP. After a time lag of 10 ms, axonemes treated with protease started sliding disintegration. Axonemes without protease digestion started nanometer-scale high-frequency oscillation after a similar time lag. Force development in the sliding disintegration was measured with a flexible glass needle and its time course was corresponded well to that of the dynein-ADP intermediate production estimated using kinetic rates previously reported. However, with a high concentration (ϳ80 M) of vanadate, which binds to the dynein-ADP intermediate and forms a stable complex of dynein-ADP-vanadate, the time course of force development in sliding disintegration was not affected at all. In the case of high frequency oscillation, the time lag to start the oscillation, the initial amplitude, and the initial frequency were not affected by vanadate, though the oscillation once started was damped more quickly at higher concentrations of vanadate. These results suggest that during the initial turnover of ATP hydrolysis, force generation of dynein is not blocked by vanadate. A vanadate-insensitive dynein-ADP is postulated as a force-generating intermediate.

the lysosomal polypeptide transporter tApL belongs to the superfamily of Atp-binding cassette transporters. tApL forms a homodimeric transport complex, which translocates oligo-and polypeptides into the lumen of lysosomes driven by Atp hydrolysis. Although the structure and the function of ABc transporters were intensively studied in the past, details about the single steps of the transport cycle are still elusive. therefore, we analyzed the coupling of peptide binding, transport and Atp hydrolysis for different substrate sizes. Although longer and shorter peptides bind with the same affinity and are transported with identical K m values, they differ significantly in their transport rates. This difference can be attributed to a higher activation energy for the longer peptide. tApL shows a basal Atpase activity, which is inhibited in the presence of longer peptides. Uncoupling between Atp hydrolysis and peptide transport increases with peptide length. Remarkably, also the type of nucleotide determines the uncoupling. While GTP is hydrolyzed as good as ATP, peptide transport is significantly reduced. In conclusion, TAPL does not differentiate between transport substrates in the binding process but during the following steps in the transport cycle, whereas, on the other hand, not only the coupling efficiency but also the activation energy varies depending on the size of peptide substrate. ATP binding cassette (ABC) transporters are part of the ABC protein superfamily and form the largest class of primary active transport proteins, which couple ATP hydrolysis with substrate transport. The substrate spectrum of ABC transporters ranges from small soluble molecules, hydrophobic drugs and lipids to large proteins 1 . ABC transporters include importers, exporters, channels, and regulators. Their basic architecture comprises two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). In eukaryotes, the ABC transporters exist as full-transporters with all four domains combined in one polypeptide chain or as half-transporters composed of one TMD and one NBD. Consequently, half-transporters can either form homo-or heterodimers. Since many transporters are linked to human diseases, such as cystic fibrosis, Tangier disease, or Dubin-Johnson syndrome, elucidating the molecular mechanisms and functions of ABC transporters is of high interest 2,3 . A representative ABC exporter is the half-transporter associated with antigen processing-like (ABCB9, TAPL), which translocates peptides into the lumen of lysosomes 4 . Besides its broad tissue distribution with highest expression in dendritic cells, macrophages, and Sertoli cells, TAPL orthologous are also found in invertebrates like Caenorhabditis elegans and even in plants . The core complex of TAPL (coreTAPL), composed of 2 × 6 C-terminal transmembrane helices (TMHs) and two NBDs, forms homodimers and is fully active in ATP hydrolysis as well as in substrate translocation. However, coreTAPL is mistargeted to the plasma membrane since lysosomal targeting is encoded by the N-terminal, four TMHs comprising TMD0 9,10 . This domain is also important for the interaction with the lysosomal membrane proteins LAMP-1/2, increasing the half-life of the transporter 11 . TAPL displays a strictly unidirectional transport of cytosolic peptides ranging from 6-up to 59-mers into the lysosomal lumen with a substrate preference for peptides with positively charged or large hydrophobic N-and C-terminal residues . TAPL can accumulate peptides more than 1000-fold against its gradient as demonstrated by dual color fluorescence burst analysis. However, peptide transport stops at a luminal concentration of 1 mM. In this trans-inhibition state, the luminal peptide potentially occupies the low affinity binding site and prevents the TMD to return back to the inward conformation 14 .

The adaptive immune system co-evolved with sophisticated pathways of antigen processing for efficient clearance of viral infections and malignant transformation. Antigenic peptides are primarily generated by proteasomal degradation and translocated into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). In the ER, peptides are loaded onto major histocompatibility complex I (MHC I) molecules orchestrated by a multisubunit peptide-loading complex (PLC). Peptide-MHC I complexes are targeted to the cell surface for antigen presentation to cytotoxic T cells, which eventually leads to the elimination of virally infected or malignantly transformed cells. Here, we review MHC I mediated antigen processing with a primary focus on the function and structural organization of the heterodimeric ATP-binding cassette (ABC) transporter TAP1/2. We discuss recent data on the molecular transport mechanism of the antigen translocation complex with respect to structural and biochemical information of other ABC exporters. We further summarize how TAP provides a scaffold for the assembly of the macromolecular PLC, thereby coupling peptide translocation with MHC I loading. TAP inhibition by distinct viral evasins highlights the important role of TAP in adaptive immunity.

Magnetic Resonance Spectroscopy affords the possibility of assessing in vivo the thermodynamic status of living tissues. The main thermodynamic variables relevant for the knowledge of the health of living tissues are: DG of ATP hydrolysis and cytosolic [ADP], the latter as calculated from the apparent equilibrium constant of the creatine kinase reaction. In this study we assessed the stoichiometric equilibrium constant of the creatine kinase reaction by in vitro 31 P NMR measurements and computer calculations resulting to be: logK CK =8.00F0.07 at T=310 K and ionic strength I=0.25 M. This value refers to the equilibrium: We also assessed by computer calculation the stoichiometric equilibrium constant of ATP hydrolysis obtaining the value: logK ATP-hyd = À12.45 at T=310 K and ionic strength I=0.25 M, which refers to the equilibrium: Finally, we formulated novel quantitative mathematical expressions of DG of ATP hydrolysis and of the apparent equilibrium constant of the creatine kinase reaction as a function of total [PCr], pH and pMg, all quantities measurable by in vivo 31 P MRS. Our novel mathematical expressions allow the in vivo assessment of cytosolic [ADP] and DG of ATP hydrolysis in the human brain and skeletal muscle taking into account pH and pMg changes occurring in living tissues both in physiological and pathological conditions.

Electrocyte membranes of Electrophorus dectricus exhibit high ATPase activity, as demonstrated by cytochemical and biochemical techniques. This activity is visualized as electrondense deposits in electron micrographs, and appears to be localized only at the innervated face of the electrocyte. AlP hydrolysis can be detected cytochemically or biochemically only in the presence of calcium or magnesium. The effects ofCa or Mg on ATPase activity can be described by Micbaelislike functions with similar apparent Km values for Ca and Mg (0.41 mM and 0.23 mM, respectively).

The properties of the mitochondrial F 1 F O -ATPase catalytic site, which can bind Mg 2+ , Mn 2+ or Ca 2+ and hydrolyses ATP, are explored by inhibition kinetic analyses to cast light on the Ca 2+ -activated F 1 F O -ATPase connection with the permeability transition pore (PTP), which initiates cascade events leading to cell death. While the natural cofactor Mg 2+ activates the F 1 F O -ATPase in competition with Mn 2+ , Ca 2+ is uncompetitive inhibitor in the presence of Mg 2+ . Selective F 1 inhibitors, namely NBD-Cl, piceatannol, resveratrol and quercetin (Is-F 1 ), exert different mechanisms (mixed/uncompetitive inhibition) on the Ca 2+ -or Mg 2+ -activated F 1 F O -ATPase, thus suggesting that the catalytic mechanism changes when Mg 2+ is replaced by Ca 2+ . In purified F 1 domain, the Ca 2+ -activated F 1 -ATPase maintains the Is-F 1 sensitivity. The enzyme inhibition is accompanied by the maintenance of the mitochondrial calcium retention capacity and membrane potential. The data strengthen the Ca 2+ -activated F 1 F O -ATPase structural relationship with the PTP, in turn involved in physiopathological cellular changes.

Myosins are essential for producing force and movement in cells through their interactions with F-actin. Generation of movement is proposed to occur through structural changes within the myosin motor domain, fuelled by ATP hydrolysis, that are amplified by a lever swing 1 , transitioning myosin from a primed (prepowerstroke) state to a post-powerstroke state. However, the initial, primed actomyosin state, proposed to form prior to lever swing, has never been observed. Nor has the mechanism by which actin catalyses myosin ATPase activity been resolved. To address this, we performed time-resolved cryoEM of a myosin-5 mutant having slow hydrolysis product release. Primed actomyosin was captured 10 ms after mixing primed myosin with F-actin, whereas postpowerstroke actomyosin predominated at 120 ms, with no abundant intermediate .

Protein dynamics is shaped by interactions between thermal fluctuations, external forces, and molecular structures. Thermal fluctuations have high frequencies and are hence very challenging to quantify. We were able to record femtosecond X-ray diffraction snapshots and determine atomic displacements of crystalline bovine trypsin atoms either in the native steady state or in a transient state initiated by a short THz pulse, the results of which were interpreted with numerical simulations. In the absence of THz pulses, specific distal atoms exhibited correlated movements. Under the influence of THz fields, numerical simulations demonstrated a slight enhancement of displacement correlations. The experimental results revealed a contrasting response to short THz pulses, where the measured displacements were not solely determined by the atom position, but also influenced by the

The small molecule EMD 57033 has been shown to stimulate the actomyosin ATPase activity and contractility of myofilaments. Here, we show that EMD 57033 binds to an allosteric pocket in the myosin motor domain. EMD 57033-binding protects myosin against heat stress and thermal denaturation. In the presence of EMD 57033, ATP hydrolysis, coupling between actin and nucleotide binding sites, and actin affinity in the presence of ATP are increased more than 10-fold. Addition of EMD 57033 to heat-inactivated β-cardiac myosin is followed by refolding and reactivation of ATPase and motile activities. In heat-stressed cardiomyocytes expression of the stress-marker atrial natriuretic peptide is suppressed by EMD 57033. Thus, EMD 57033 displays a much wider spectrum of activities than those previously associated with small, drug-like compounds. Allosteric effectors that mediate refolding and enhance enzymatic function have the potential to improve the treatment of heart failure, myopathies, and protein misfolding diseases.

The Escherichia coli Rep helicase is a dimeric motor protein that catalyzes the transient unwinding of duplex DNA to form single-stranded (ss) DNA using energy derived from the binding and hydrolysis of ATP. In an effort to understand this mechanism of energy transduction, we have used pre-steady-state methods to study the kinetics of ATP binding and hydrolysis by an important intermediate in the DNA unwinding reactionsthe asymmetric Rep dimer state, P 2 S, where ss DNA [dT(pT) 15 ] is bound to only one subunit of the Rep dimer. To differentiate between the two potential ATPase active sites inherent in the dimer, we constructed dimers with one subunit covalently cross-linked to ss DNA and where one or the other of the ATPase sites was selectively complexed to the tightly bound transition state analog ADP-AlF 4 . We found that when ADP-AlF 4 is bound to the Rep subunit in trans from the subunit bound to ss DNA, steady-state ATPase activity of 18 s -1 per dimer (equivalent to wild-type P 2 S) was recovered. However, when the ADP-AlF 4 and ss DNA are both bound to the same subunit (cis), then a titratable burst of ATP hydrolysis is observed corresponding to a single turnover of ATP. Rapid chemical quenched-flow techniques were used to resolve the following minimal mechanism for ATP hydrolysis by the unligated Rep subunit of the cis dimer: .0 ( 0.2 mM, and K 6 ) 80 ( 8 µM. A salient feature of this mechanism is the presence of a kinetically trapped long-lived tight nucleotide binding state, E′-ADP-P i . In the context of our "subunit switching" model for Rep dimer translocation during processive DNA unwinding [Bjornson, K. B., Wong, I., & Lohman, T. M. (1996) J. Mol. Biol. 263,[411][412][413][414][415][416][417][418][419][420][421][422], this state may serve an energy storage function, allowing the energy from the binding and hydrolysis of ATP to be harnessed and held in reserve for DNA unwinding.

Myosin activation is a viable approach to treat systolic heart failure. We previously demonstrated that striated muscle myosin is a promiscuous ATPase that can use most nucleoside triphosphates as energy substrates for contraction. When 2-deoxy ATP (dATP) is used, it acts as a myosin activator, enhancing cross-bridge binding and cycling. In vivo, we have demonstrated that elevated dATP levels increase basal cardiac function and rescues function of infarcted rodent and pig hearts. Here we investigate the molecular mechanism underlying this physiological effect. We show with molecular dynamics simulations that the binding of dADP.Pi (dATP hydrolysis products) to myosin alters the structure and dynamics of the nucleotide binding pocket, myosin cleft conformation, and actin binding sites, which collectively yield a myosin conformation that we predict favors weak, electrostatic binding to actin. In vitro motility assays at high ionic strength were conducted to test this prediction and we...

Here we characterized this conformation using pure mouse MDR3 P-glycoprotein and natural MgATP and MgADP. Mutants E552A/E1197A, E552Q/E1197Q, E552D/ E1197D, and E552K/E1197K had low but real ATPase activity in the order Ala > Gln > Asp > Lys, emphasizing the requirement for Glu stereochemistry. Mutant E552A/ E1197A bound MgATP and MgADP (1 mol/mol) with K d 9.2 and 92 M, showed strong temperature sensitivity of MgATP binding and equal dissociation rates for MgATP and MgADP. With MgATP as the added ligand, 80% of bound nucleotide was in the form of ATP. None of these parameters was vanadate-sensitive. The other mutants showed lower stoichiometry of MgATP and MgADP binding, in the order Ala > Gln > Asp > Lys. We conclude that the E552A/E1197A mutation arrests the enzyme in a conformation, likely a stabilized NBD dimer, which occludes nucleotide, shows preferential binding of ATP, does not progress to a normal vanadate-sensitive transition state, but hydrolyzes ATP and releases ADP slowly. Impairment of turnover is primarily due to inability to form the normal transition state rather than to slow ADP release. The Gln, Asp, and Lys mutants are less effective at stabilizing the occluded nucleotide, putative dimeric NBD, conformation. We envisage that in wild-type the occluded nucleotide conformation occurs transiently after MgATP binds to both NBDs with associated dimerization, and before progression to the transition state.

Adenosine nucleotides affect the ability of RecA⅐singlestranded DNA (ssDNA) nucleoprotein filaments to cooperatively assume and maintain an extended structure that facilitates DNA pairing during recombination. Here we have determined that ADP and ATP/ATP␥S affect the DNA binding and aggregation properties of the human RecA homolog human RAD51 protein (hRAD51). These studies have revealed significant differences between hRAD51 and RecA. In the presence of ATP␥S, RecA forms a stable complex with ssDNA, while the hRAD51 ssDNA complex is destabilized. Conversely, in the presence of ADP and ATP, the RecA ssDNA complex is unstable, while the hRAD51 ssDNA complex is stabilized. We identified two hRAD51⅐ssDNA binding forms by gel shift analysis, which were distinct from a well defined RecA⅐ssDNA binding form. The available evidence suggests that a low molecular weight hRAD51⅐ssDNA binding form (hRAD51⅐ssDNA low ) correlates with active ADP and ATP processing. A high molecular weight hRAD51⅐ ssDNA aggregate (hRAD51⅐ssDNA high ) appears to correlate with a form that fails to process ADP and ATP. Our data are consistent with the notion that hRAD51 is unable to appropriately coordinate ssDNA binding with adenosine nucleotide processing. These observations suggest that other factors may assist hRAD51 in order to mirror RecA recombinational function.

Here we characterized this conformation using pure mouse MDR3 P-glycoprotein and natural MgATP and MgADP. Mutants E552A/E1197A, E552Q/E1197Q, E552D/ E1197D, and E552K/E1197K had low but real ATPase activity in the order Ala > Gln > Asp > Lys, emphasizing the requirement for Glu stereochemistry. Mutant E552A/ E1197A bound MgATP and MgADP (1 mol/mol) with K d 9.2 and 92 M, showed strong temperature sensitivity of MgATP binding and equal dissociation rates for MgATP and MgADP. With MgATP as the added ligand, 80% of bound nucleotide was in the form of ATP. None of these parameters was vanadate-sensitive. The other mutants showed lower stoichiometry of MgATP and MgADP binding, in the order Ala > Gln > Asp > Lys. We conclude that the E552A/E1197A mutation arrests the enzyme in a conformation, likely a stabilized NBD dimer, which occludes nucleotide, shows preferential binding of ATP, does not progress to a normal vanadate-sensitive transition state, but hydrolyzes ATP and releases ADP slowly. Impairment of turnover is primarily due to inability to form the normal transition state rather than to slow ADP release. The Gln, Asp, and Lys mutants are less effective at stabilizing the occluded nucleotide, putative dimeric NBD, conformation. We envisage that in wild-type the occluded nucleotide conformation occurs transiently after MgATP binds to both NBDs with associated dimerization, and before progression to the transition state.

Ecto-nucleoside triphosphate diphosphohydrolases, NTPDase1 (CD39) and NTPDase3, are integral plasma membrane proteins that hydrolyze extracellular nucleotides, thereby modulating the function of purinergic receptors. During processing in the secretory pathway, the active sites of ectonucleotidases are located in the lumen of vesicular compartments, thus raising the question whether the ecto-nucleotidases affect the ATP-dependent processes in these compartments, including protein folding in the endoplasmic reticulum (ER). It has been reported that CD39 is not active until it reaches the plasma membrane, suggesting that terminal glycosylation in Golgi is critical for its activity. To investigate the subcellular location and the mechanism of ecto-nucleotidase activation, we expressed human NTPDase3 in COS-1 cells and blocked the secretory transport with monensin or brefeldin A, or by targeting to ER with a signal peptide. Cell surface biotinylation, sensitivity to glycosidases, and fluorescence microscopy analyses suggest that, in contrast to the previous report on CD39, NTPDase3 becomes catalytically active in the ER or in the ER-Golgi intermediate compartment, and that terminal glycosylation in Golgi is not essential for activity. Moreover, ER-targeted NTPDase3, but not wild-type NTPDase3 or ER-targeted inactive G221A mutant, significantly diminished the folding efficiency and the transport to the plasma membrane of co-expressed CD39 used as a reporter protein. These data suggest that ER-targeted NTPDase3 significantly depletes ATP in ER, whereas wild-type NTPDase3 is likely to acquire ATPase activity in a post-ER, but pre-Golgi, compartment, thus avoiding unproductive ATP hydrolysis and interference with protein folding in the ER. ERtargeted NTPDase3 may be a useful experimental tool to study the effects of ER ATP depletion on ER function under normal and stress conditions.

DNA can be denatured by two main methods which are: a) denaturation in solution (invitro) and b) denaturation on a slide surface (in-situ). Additionally, DNA can also be denatured in gels with urea. The method to be used depends on various factors such as the application, the source of the DNA, the length, and the techniques available to confirm the extent of denaturation. Verification of the extent of denaturation is important because of the following factors: 1) increases the chances of hybridization (especially for short probes), 2) prevents the loss of expensive probes (if the target site is not denatured then, the probes will not hybridize and will only cause a high a background), 3) a higher degree of denaturation allows for more probes to be used and therefore, more information can be derived after hybridization, and 4) essential to maximize due to extremely short probe length. It is important to ensure that DNA morphology is preserved after denaturation in order for the prob...

Cohesin’s association with and translocation along chromosomal DNAs depend on an ATP hydrolysis cycle driving the association and subsequent release of DNA. This involves DNA being ‘clamped’ by Scc2 and ATP-dependent engagement of cohesin’s Smc1 and Smc3 head domains. Scc2’s replacement by Pds5 abrogates cohesin’s ATPase and has an important role in halting DNA loop extrusion. The ATPase domains of all SMC proteins are separated from their hinge dimerisation domains by 50-nm-long coiled coils, which have been observed to zip up along their entire length and fold around an elbow, thereby greatly shortening the distance between hinges and ATPase heads. Whether folding exists in vivo or has any physiological importance is not known. We present here a cryo-EM structure of the apo form of cohesin that reveals the structure of folded and zipped-up coils in unprecedented detail and shows that Scc2 can associate with Smc1’s ATPase head even when it is fully disengaged from that of Smc3. Usi...

Smc–ScpAB forms elongated, annular structures that promote chromosome segregation, presumably by compacting and resolving sister DNA molecules. The mechanistic basis for its action, however, is only poorly understood. Here, we have established a physical assay to determine whether the binding of condensin to native chromosomes in Bacillus subtilis involves entrapment of DNA by the Smc–ScpAB ring. To do so, we have chemically cross-linked the three ring interfaces in Smc–ScpAB and thereafter isolated intact chromosomes under protein denaturing conditions. Exclusively species of Smc–ScpA, which were previously cross-linked into covalent rings, remained associated with chromosomal DNA. DNA entrapment is abolished by mutations that interfere with the Smc ATPase cycle and strongly reduced when the recruitment factor ParB is deleted, implying that most Smc–ScpAB is loaded onto the chromosome at parS sites near the replication origin. We furthermore report a physical interaction between na...

The vacuolar ATPase enzyme complex (V-ATPase) pumps protons across membranes, energised by hydrolysis of ATP. It is involved in many physiological processes and has been implicated in many different diseases. While the broader functions of V-ATPases have been reviewed extensively, the role of this complex in nematodes specifically has not. Here, the essential role of the V-ATPase in nematode nutrition, osmoregulation, synthesis of the cuticle, neurobiology and reproduction is discussed. Based on the requirement of V-ATPase activity, or components of the V-ATPase, for these processes, the potential of the V-ATPase as a drug target for nematode parasites, which cause a significant burden to human health and agriculture, is also discussed. The V-ATPase has all the characteristics of a suitable drug target against nematodes, however the challenge will be to develop a high-throughput assay with which to test potential inhibitors.

Disruption of the gene encoding RAD51, the protein that catalyzes strand exchange during homologous recombination, leads to the accumulation of chromosome breaks and lethality in vertebrate cells. As RAD51 is implicated in BRCA1-and BRCA2-mediated tumor suppression as well as cellular viability, we have begun a functional analysis of a defined RAD51 mutation in mammalian cells. By using a dominant negative approach, we generated a mouse embryonic stem cell line that expresses an ATP hydrolysis-defective RAD51 protein, hRAD51-K133R, at comparable levels to the endogenous wild-type RAD51 protein, whose expression is retained in these cells. We found that these cells have increased sensitivity to the DNA-damaging agents mitomycin C and ionizing radiation and also exhibit a decreased rate of spontaneous sister-chromatid exchange. By using a reporter for the repair of a single chromosomal double-strand break, we also found that expression of the hRAD51-K133R protein specifically inhibits homology-directed double-strand break repair. Furthermore, expression of a BRC repeat from BRCA2, a peptide inhibitor of an early step necessary for strand exchange, exacerbates the inhibition of homology-directed repair in the hRAD51-K133R expressing cell line. Thus, ATP hydrolysis by RAD51 has a key role in various types of DNA repair in mammalian cells.

Disruption of the gene encoding RAD51, the protein that catalyzes strand exchange during homologous recombination, leads to the accumulation of chromosome breaks and lethality in vertebrate cells. As RAD51 is implicated in BRCA1-and BRCA2-mediated tumor suppression as well as cellular viability, we have begun a functional analysis of a defined RAD51 mutation in mammalian cells. By using a dominant negative approach, we generated a mouse embryonic stem cell line that expresses an ATP hydrolysis-defective RAD51 protein, hRAD51-K133R, at comparable levels to the endogenous wild-type RAD51 protein, whose expression is retained in these cells. We found that these cells have increased sensitivity to the DNA-damaging agents mitomycin C and ionizing radiation and also exhibit a decreased rate of spontaneous sister-chromatid exchange. By using a reporter for the repair of a single chromosomal double-strand break, we also found that expression of the hRAD51-K133R protein specifically inhibits homology-directed double-strand break repair. Furthermore, expression of a BRC repeat from BRCA2, a peptide inhibitor of an early step necessary for strand exchange, exacerbates the inhibition of homology-directed repair in the hRAD51-K133R expressing cell line. Thus, ATP hydrolysis by RAD51 has a key role in various types of DNA repair in mammalian cells.

Disruption of the gene encoding RAD51, the protein that catalyzes strand exchange during homologous recombination, leads to the accumulation of chromosome breaks and lethality in vertebrate cells. As RAD51 is implicated in BRCA1-and BRCA2-mediated tumor suppression as well as cellular viability, we have begun a functional analysis of a defined RAD51 mutation in mammalian cells. By using a dominant negative approach, we generated a mouse embryonic stem cell line that expresses an ATP hydrolysis-defective RAD51 protein, hRAD51-K133R, at comparable levels to the endogenous wild-type RAD51 protein, whose expression is retained in these cells. We found that these cells have increased sensitivity to the DNA-damaging agents mitomycin C and ionizing radiation and also exhibit a decreased rate of spontaneous sister-chromatid exchange. By using a reporter for the repair of a single chromosomal double-strand break, we also found that expression of the hRAD51-K133R protein specifically inhibits homology-directed double-strand break repair. Furthermore, expression of a BRC repeat from BRCA2, a peptide inhibitor of an early step necessary for strand exchange, exacerbates the inhibition of homology-directed repair in the hRAD51-K133R expressing cell line. Thus, ATP hydrolysis by RAD51 has a key role in various types of DNA repair in mammalian cells.

Cytokinesis in Gram-negative bacteria requires coordinated invagination of the three layers of the cell envelope; otherwise, cells become sensitive to hydrophobic antibiotics and can even undergo cell lysis. In E. coli , the ABC transporter FtsEX couples the synthesis and hydrolysis of the stress-bearing peptidoglycan layer at the septum by interacting with FtsA and EnvC, respectively. ATP hydrolysis by FtsEX is critical for its function, but the reason why is not clear. Here, we find that in the absence of ATP hydrolysis, FtsEX blocks septal PG synthesis similarly to cephalexin. However, an FtsEX ATPase mutant, under conditions where it cannot block division, rescues ftsEX phenotypes as long as a partially redundant cell separation system is present. Furthermore, we find that the FtsEX-FtsA interaction is important for efficient cell separation.

ABC transporters form one of the largest and ancient of protein families. ABC transporters 3 couple hydrolysis of ATP to vectorial translocation of diverse substrates across cellular 4 membranes. Many human ABC transporters are medically important in causing, for example, 5 multidrug resistance to cytotoxic drugs. Seven complete prokaryotic structures and one 6 eukaryotic structure were solved for transporters from 2002 to the present, and a wealth of 7 research is being conducted on and around these structures in order to resolve the mechanistic 8 conundrum of how these transporters couple ATP hydrolysis in cytosolic domains to substrate 9 translocation through the transmembrane pore. Many questions remained unanswered about 10 this mechanism, despite a plethora of data and a number of interesting and controversial 11 models. 12 13 14 15 ATP-Binding Cassette (ABC) transporters are found in all phyla and constitute one of the 4 largest protein superfamilies [1] and [2]. A recent analysis of the full genomes from 13 diverse 5 organisms from the kingdoms, archaea, eubacteria, and eukarya identified sequences within 6 genes encoding the nucleotide-binding domains of ABC transporters as amongst the most 7 conserved phylogenic DNA sequences [3]. ABC transporters couple hydrolysis of ATP to 8 vectorial translocation of substrates across cellular membranes, typically against a 9 concentration gradient. Through their transport function these integral membrane proteins are 10 involved in diverse cellular processes such as maintenance of osmotic homeostasis, nutrient 11 uptake, resistance to cytotoxic drugs and antibiotics, cell division, bacterial immunity, 12 pathogenesis and sporulation, cholesterol and lipid trafficking, cellular immune response, and 13 developmental stem cell biology [4] and [5]. Many of the human ABC transporters are 14 medically important, including ABCC7/CFTR that causes cystic fibrosis by any of dozens of 15 mutations in the gene. A subclass of ABC transporters are associated with multidrug resistance, 16 through the extrusion of cytotoxic agents used in chemotherapy against tumours. ABCB1/P-17 glycoptotein/MDR1, ABCC1/MRP1, and ABCG2/BCRP ABC transporters appear to account 18 for nearly all of the MDR tumour cells in both humans and rodents. All of the medically 19 important ABC transporters can be located at: ; 20 and . 21 Look at how they are built: different peas in the same pod? nucleotide-binding domains (NBDs). The four domains may be comprised of one, two or four 1 polypeptide chains, encoded by the same or different genes, which assemble into monomers, 2 homo-or heterodimers, or tetramers. Prokaryotes harbour both importers for nutrient uptake 3 (including amino acids, sugars, metal ions) and exporters (drugs, toxins, polysaccharides, 4 lipids, proteins), and eukaryotes only exporters [6] and [7]. For example, the K12 serotype of E. 5 coli contains 65 experimentally verified and putative ABC transporters. Of the 65 ABC 6 transporters listed, 50 are ABC importers and the remaining 15 are exporters [8]. Bacterial 7 ABC importers also contain a periplasmic binding protein that captures substrate and delivers it 8 to the transporter. The TMDs, which contain cytosolic as well as membrane spanning regions, 9 form the transmembrane (TM) pore and contain the substrate binding sites, while the NBDs 10 bind and hydrolyse ATP. The cytosolic regions of the TMDs, known as the intracytoplasmic 11 loops (ICLs), form the physical interface between the TMDs and NBDs and are thought to 12 coordinate ATP binding and hydrolysis with substrate binding and translocation. Sequence 13 analysis indicates that the consensus configuration of the TMDs is of two sets of six 14 hydrophobic TM spans, though there are notable exceptions among importers, with some 15 transporters having ten or more while others have fewer than six. The TMDs are thus not well 16 conserved in length or sequence, probably reflecting their role in binding diverse substrates. In 17 a functional ABC transporter, two TMDs form a selectively permeable pore or conduit through 18 the membrane. At any point in time, the TMDs are "gated" so that they are closed to one side 19 of the membrane, preventing passive diffusion of substrates. 20 21 Each NBD is roughly an L-shape with two lobes comprising three subdomains. The larger 22 Lobe I includes a RecA-and F 1 -ATPase-like ATP-binding core subdomain [9] (Figure , blue), 23 containing the Walker A (GXXGXGKS/T) and B (DE) motifs, where '' is any aliphatic

E. coli ClpB is a heat shock protein that belongs to the AAAþ protein family. Studies have shown that ClpB and its eukaryotic homologue, Hsp104, can disaggregate denatured proteins by themselves or cooperate with the DnaK chaperone system in vivo. It is thought that ClpB requires binding of nucleoside triphosphate to assemble into hexameric rings with protein binding activity and ClpB majorly exist as hexamer in the presence of nucleoside triphosphate. In contrast to this conclusion, our sedimentation velocity data show that ClpB can form hexamer in the absence of nucleotide and ClpB resides in a monomer-dimer-tetramer-hexamer equilibrium in the presence of ATPgS (a slowly hydrolysable ATP analog). ClpB hexamers exhibit fast subunits exchange in the absence of nucleoside triphosphate, while the exchange rates decrease in the presence of a large excess of ATPgS. We anticipate our studies on ClpB assembly to be a starting point of for understanding how ClpB hexamers disaggregate protein aggregates. For example, knowing the population of ClpB hexamers in solution is essential for the interpretation of it is only possible to study the energetics and kinetics data for the of ClpB catalyzed disaggregation processreaction by knowing the population of ClpB hexamer in solution.

P-glycoprotein (P-gp or ABCB1) is a member of the broad family of ABC transporters. P-gp participates in the establishment of physiological barriers limiting cellular access of a large number of toxic compounds. It thus plays important roles in the pharmacokinetics of these compounds. Cancer cells and cells infected by viruses exploit the presence of P-gp to fend off drug treatment, rendering them multidrug-resistant. Overcoming multidrug resistance caused by expression of ABC transporters has gained increasing attention in the field of drug development. Recently, studies of P-gp, especially from structural investigations by both cryo-electron microscopy and X-ray crystallography, have provided high-resolution mechanistic details for the function of this transporter. Structures with increasing resolution and accuracy in various substrate-and inhibitor-bound forms are available for analysis and a consensus on the mechanism of substrate polyspecificity is emerging. The use of new structural information may aid development of P-gp inhibitors as well as compounds that may bypass P-gp action.

The human ABC (ATP-binding cassette) transporter MRP1 (human multidrug-resistance-associated protein 1; ABCC1) is involved in the cellular extrusion of conjugated metabolites and causes multidrug resistance in tumour cells. The transport of substrate molecules by ABC proteins is energized by ATP hydrolysis, performed by two co-operating ABC units. Orthovanadate (Vi), a non-covalent inhibitor of the ABC ATPases, was found to catalyse a photo-oxidative cleavage of various ATP-binding proteins. In the present study, we have identified three Vi-cleavage sites within MRP1, and found that the cleavage reactions were variably modulated by the presence of nucleotides and by transported substrates. We concluded that Vi cleavage of MRP1 at Site I detects conformational changes due to the binding of MgATP. In contrast, Site II could be identified as part of the substrate-modulated catalytic cycle, probably containing an MRP1·MgADP·Vi transition-state-like complex. Cleavage at Site III was modu...

Hsp70 chaperones play important roles in cells including protein folding, trafficking, degradation and enabling survival under stress conditions. DnaK is an E. coli Hsp70 homolog comprising an ATPase domain and a substrate-binding domain. DnaK has two substrate-affinity states: ATP binding lowers the affinity of the substrate, whereas its hydrolysis leads to a higher affinity of substrate for binding. ATP-dependent communication between the two domains is essential for chaperone function and mediated via a conserved hydrophobic linker ( 384 GDVKDVLLL 392 ). Previous studies showed that when the linker interacts with the ATPase domain, which was studied by the construct containing the entire linker, DnaK(1-392), an enhanced ATPase rate is observed compared to the construct lacking the conserved 389 VLLL 392 linker region, DnaK(1-388). This observation suggests that structural rearrangements caused by linker docking adopt the ATPase domain in a closed conformation, leading to an enhanced, pH-dependent ATPase activity. Here, our aim is to delineate the residues that are responsible for the linker induced conformational rearrangements. In that line, using molecular dynamic simulations we identified a putative network of interactions through Arg71-Glu81-Asp85-Thr225-His226 at the lobe interface of the ATPase domain that might be critical in stabilization of the domain in the so called ''open'' and ''closed'' conformational equilibrium. We made point mutations for these sites on the two ATPase domain constructs, and studied the structural and functional effects of these residues on the ATPase domain as a function of pH using various biophysical and biochemical methods. Mutations' effects studied by equilibrium thermodynamic measurements showed variations for the constructs, but dramatic changes were observed in the dynamics of the constructs. Our results suggest the linker as a controller for ATPase domain dynamics changes partly through the identified network.

The inhibition of F~-ATPase by its natural peptide inhibitor is mixed non-competitive with two pH optimum values (5.5 and 8.2). 2. A two-step model for the interaction is suggested in which two enzyme conformations would exhibit different affinities for the peptide. 3. At low pH. interaction would be favoured. At high pH, a conformation (not susceptible to inhibitionJ changes into another (susceptible to inhibition) through the hydrolytic reaction stimulation, due to high pH.

Release of the ATP hydrolysis product inorganic phosphate (Pi) from the active site of myosin is central in chemo-mechanical energy transduction and closely associated with the main force-generating structural change, the power-stroke. Despite intense investigations, the relative timing between Pi-release and the power-stroke remains poorly understood. This hampers in depth understanding of the production of force and motion by myosin in health and disease and also our understanding of myosin-active drugs. From the 1990s and up to today, models with the Pi-release either distinctly before or after the power-stroke, in unbranched kinetic schemes, have dominated the literature. However, in recent years, alternative models have emerged to explain apparently contradictory findings. Here, we first compare and critically analyze, three influential alternative models, either characterized by a branched kinetic scheme or by partial uncoupling of Pi-release and the power-stroke. Finally, we ...

In Escherichia coli, FtsEX coordinates peptidoglycan (PG) synthesis and hydrolysis at the septum. It acts on FtsA in the cytoplasm to promote recruitment of septal PG synthetases and recruits EnvC, an activator of septal PG hydrolases, in the periplasm. Following recruitment, ATP hydrolysis by FtsEX is thought to regulate both PG synthesis and hydrolysis, but how it does this is not well understood. Here, we show that an ATPase mutant of FtsEX blocks septal PG synthesis similarly to cephalexin, suggesting that ATP hydrolysis by FtsEX is required throughout septation. Using mutants that uncouple the roles of FtsEX in septal PG synthesis and hydrolysis, we find that recruitment of EnvC to the septum by FtsEX, but not ATP hydrolysis, is required to promote cell separation when the NlpD-mediated cell separation system is present. However, ATP hydrolysis by FtsEX becomes necessary for efficient cell separation when the NlpD system is inactivated, suggesting that the ATPase activity of FtsEX is required for optimal activity of EnvC. Importantly, under conditions that suppress the role of FtsEX in cell division, disruption of the FtsEX-FtsA interaction delays cell separation, highlighting the importance of this interaction in coupling the cell separation system with the septal PG synthetic complex. IMPORTANCE Cytokinesis in Gram-negative bacteria requires coordinated invagination of the three layers of the cell envelope; otherwise, cells become sensitive to hydrophobic antibiotics and can even undergo cell lysis. In E. coli, the ABC transporter FtsEX couples the synthesis and hydrolysis of the stress-bearing peptidoglycan layer at the septum by interacting with FtsA and EnvC, respectively. ATP hydrolysis by Ft-sEX is critical for its function, but the reason why is not clear. Here, we find that in the absence of ATP hydrolysis, FtsEX blocks septal PG synthesis similarly to cephalexin. However, an FtsEX ATPase mutant, under conditions where it cannot block division, rescues ftsEX phenotypes as long as a partially redundant cell separation system is present. Furthermore, we find that the FtsEX-FtsA interaction is important for efficient cell separation.

Cytokinesis in Gram-negative bacteria requires coordinated invagination of the three layers of the cell envelope; otherwise, cells become sensitive to hydrophobic antibiotics and can even undergo cell lysis. In E. coli , the ABC transporter FtsEX couples the synthesis and hydrolysis of the stress-bearing peptidoglycan layer at the septum by interacting with FtsA and EnvC, respectively. ATP hydrolysis by FtsEX is critical for its function, but the reason why is not clear. Here, we find that in the absence of ATP hydrolysis, FtsEX blocks septal PG synthesis similarly to cephalexin. However, an FtsEX ATPase mutant, under conditions where it cannot block division, rescues ftsEX phenotypes as long as a partially redundant cell separation system is present. Furthermore, we find that the FtsEX-FtsA interaction is important for efficient cell separation.

The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (...

through diverse mechanisms that converge in the activation of PGC-1α, leading to enhancing transcriptional activity and mitochondrial remodeling. Micro-RNAs (miRNAs) have been known to act as powerful negative modulators of gene expressions involved in essential cellular processes. Recent evidence suggests that miR-494, miR-696, and miR -761 are involved in mitochondrial biogenesis by negative modulation of PGC-1α signaling. However, it remains unclear whether these miRNAs are regulated individually or cooperatively by nutrients stimulation. Therefore, our study was focused on the effect of Leu and CAFF on these miRNAs functions and how it affected its downstream effectors, and ultimately, mitochondrial biogenesis. METHODS: Following 5 days of differentiation period, C2C12 myotubes were treated with Leu (1 and 3mM) or CAFF (3mM) containing Dulbecco's modified Eagle's medium (DMEM) without serum and Leu for 24h. The serum and Leu-Free DMEM with a 2% H 2 0 was used as a control. After 24h of each treatment, the cells were harvested and then, DNA, RNA, and protein (whole fraction) were isolated for immunoblotting and qPCR analyses. Micrographs of four fields per condition were randomly captured before and 24h after treatment to measure myotube diameter. RESULTS: Mitochondrial DNA copy number increased significantly 24h after Leu addition, and especially in the CAFF treatment (p < 0.05), compared with the control cells. Similarly, myotube diameter was significantly larger in two Leu-treated groups (≥ 20%, p < 0.05), as well as CAFF supplementation (~10%, p < 0.05), than in control and pre-treatment groups. PGC-1α protein level and phosphorylation rate of p70S6K were also augmented in both treatment groups. Conversely, miR-494, miR-696, and miR-761 levels were downregulated in the Leu-treated groups, but only miR-761 levels were decreased in the CAFF-treated group. CONCLUSION: These results suggested that nutrients such as Leu or CAFF can regulate the expression of PGC-1α-targeting miRNAs, which then leads to inducing mitochondrial biogenesis.

Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone's active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators. Protein functions are determined by their internal dynamics and are fine-tuned by the interactions with effectors of different chemical origins . Most chemical interventions on proteins and enzymes focus on the identification of small molecule inhibitors designed to abrogate their activities 4 , or on the development of agonists that induce active-like conformations of the target and subsequent signaling responses by binding to known orthosteric sites . In contrast, the rational search for allosteric activators of protein functions is still relatively unexplored 6,7 . However, allosteric activators may provide new opportunities to develop chemical tools to probe the role of protein activity in cellular phenotypes: indeed new information can be gained by turning on a signaling pathway starting from a specific node of the underlying protein network . Allosteric activators can in principle modulate receptor function while still allowing the possibility of orthosteric agonist or antagonist binding. In this context, allosteric ligands can act as affinity modulators by changing the affinity of the orthosteric ligand for the receptor, as well as induce conformational changes that in turn modulate the efficacy of the orthosteric ligand in determining cellular responses. Moreover, activators can help identify and characterize allosteric sites and mechanisms for the discovery of novel drug candidates: indeed, since allostery represents one of the most relevant means to regulate protein function, drug discovery can be extended to target regulatory protein pockets. It has previously been shown that sequence variation in allosteric pockets could aid the design of highly specific drugs that would bind only specific members of a protein family. Finally, allosteric ligands can potentially modulate the balance between the target protein conformations that are presented to other interacting proteins, generating the possibility to perturb protein-protein interactions (PPIs) through low molecular weight ligands 9,10 .

The glucocorticoid receptor (GR), like many signaling proteins requires Hsp90 for sustained activity. Previous biochemical studies revealed that the requirement for Hsp90 is explained by its ability to reverse Hsp70-mediated inactivation of GR through a complex process requiring both cochaperones and Hsp90 ATP hydrolysis. How ATP hydrolysis on Hsp90 enables GR reactivation is unknown. The canonical mechanism of client release from Hsp70 requires ADP:ATP exchange, which is normally rate limiting. Here we show that independent of ATP hydrolysis, Hsp90 acts as an Hsp70 nucleotide exchange factor (NEF) to accelerate ADP dissociation, likely coordinating GR transfer from Hsp70 to Hsp90. As Bag-1 is a canonical Hsp70 NEF that can also reactivate Hsp70:GR, the impact of these two NEFs was compared. Simple acceleration of Hsp70:GR release was insufficient for GR reactivation as Hsp70 rapidly re-binds and re-inactivates GR. Instead, inhibition of GR re-inactivation by Hsp70 is critical. This...

P 1B -type ATPases are polytopic membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic transition metals. This article reviews recent progress in our understanding of the structure and function of these proteins in bacteria. These are members of the P-type superfamily of transport ATPases. Cu + -ATPases are the most frequently observed and bestcharacterized members of this group of transporters. However, bacterial genomes show diverse arrays of P 1B -type ATPases with a range of substrates (Cu+, Zn2+, Co2+). Furthermore, because of the structural similarities among transitions metals, these proteins can also transport nonphysiological substrates (Cu 2+ , Cd 2+ , Pb 2+ , Au + , Ag + ). P 1B -type ATPases have six or eight transmembrane segments (TM) with metal coordinating amino acids in three core TMs flanking the cytoplasmic domain responsible for ATP binding and hydrolysis. In addition, regulatory cytoplasmic metal binding domains are present in most P 1B -type ATPases. Central to the transport mechanism is the binding of the uncomplexed metal to these proteins when cytoplasmic substrates are bound to chaperone and chelating molecules. Metal binding to regulatory sites is through a reversible metal exchange among chaperones and cytoplasmic metal binding domains. In contrast, the chaperone-mediated metal delivery to transport sites appears as a largely irreversible event. P 1B -ATPases have two overarching physiological functions: to maintain cytoplasmic metal levels and to provide metals for the periplasmic assembly of metalloproteins. Recent studies have shown that both roles are critical for bacterial virulence, since P 1B -ATPases appear key to overcome high phagosomal metal levels and are required for the assembly of periplasmic and secreted metalloproteins that are essential for survival in extreme oxidant environments.

14‐3‐3 Proteins bind phosphorylated sequences in proteins and regulate multiple cellular functions. For the first time, we show that pure recombinant human 14‐3‐3 ζ, γ, ε and τ isofoms hydrolyze ATP with similar K m and k cat values. In sharp contrast the sigma isoform has no detectable activity. Docking studies identify two putative binding pockets in 14‐3‐3 zeta. Mutation of D124A in the amphipathic pocket enhances binding affinity and catalysis. Mutation of a critical Arg (R55A) at the dimer interface in zeta reduces binding and decreases catalysis. These experimental results coincide with a binding pose at the dimer interface. This newly identified function could be a moon lighting function in some of these isoforms.

In most tissues the mitochondrial ATP-synthase plays a central role by synthesizing the bulk of ATP. According to the classical theory of respiratory control, flux through this enzyme is solely determined by substrate (ADP) concentration while the enzyme has a fixed capacity. However, in different cell types such as rat cardiomyocytes and neurons, dog heart, human fibroblasts, and skeletal and heart muscle from children, it has been shown that active regulation of the mitochondrial ATP-synthase in response to cellular energy demand exists. For example, in rat cardiomyocytes the mitochondrial ATP-synthase activity is down-regulated in response to anoxia or mitochondrial uncoupling. By this mechanism cellular ATP is conserved, as under these conditions the ATP-synthase would work in reverse and hydrolyze ATP. When cardiomyocytes are stimulated to contract, ATP-synthase activity is up-regulated in line with the increased energy demand. Preincubation of the cardiomyocytes with positive inotropic substances results in further up-regulation of the ATP-synthase. By blocking calcium transport, it has been shown that the up-regulation of the enzyme is calcium-dependent. On a molecular level, up-regulation is probably mediated by the calcium-binding inhibitor protein (CaBI) and down-regulation via the inhibitor protein IF 1 . The ATP-synthase system is disturbed under several pathophysiological conditions. First, mutations can cause a primary defect in the mitochondrial ATP-synthase (respiratory chain defect). Furthermore, secondary defects of the ATP-synthase occur. In rat models abnormalities of ATP-synthase can be detected in different types of cardiomyopathy/heart hypertrophy. The changes are reversible in response to treatment of the heart diseases. Abnormalities of the ATPsynthase system can be observed in fibroblasts from patients with neuronal ceroidlipofuscinoses. Toxic metabolites accumulating in methylmalonic acidurias can inhibit ATP-synthase. When neurons are incubated with 3-OH glutarate-a substance accumulating in glutaric aciduria I-as a model for glutaric aciduria I, ATP-synthase activity is compromised. This lack of energy may lead to Ôslow onsetÕ excitotoxicity and finally cell death. Cells can be rescued by adding creatine to the incubation medium. In D D -2-hydroxyglutaric aciduria, inhibition of the ATP-synthase has been observed.

Background : Human Rad51 protein (HsRad51) is a homologue of Escherichia coli RecA protein, and involved in homologous recombination. These eukaryotic and bacterial proteins catalyse strand exchange between two homologous DNA molecules, each forming a complex with single-stranded DNA (ssDNA) and ATP as the initial step. Both proteins hydrolyse ATP; however, the role of ATP hydrolysis appears to vary between the two proteins. Results : Measurements using the fluorescence ssDNA analogue, poly(1, N 6 -etheno-deoxyadenosine), indicate that ATP affects the HsRad51-ssDNA complex, promoting two conformational states: one transient, rather rigid transition state and a final more flexible state. While ADP lowers the affinity of RecA protein to ssDNA, it is found to rather stabilize the HsRad51-ssDNA complex. ADP does not activate the strand exchange by HsRad51 but instead stimulates annealing between complementary ssDNAs. Conclusions : The hydrolysis of ATP promotes a transition of the HsRad51-ssDNA complex from a stiff state to less stiff state. The first state may be important for the strand separation of dsDNA in the initial step of strand exchange, while the second state may be important for annealing in the next step. However, hydrolysis does not dissociate HsRad51 from DNA as a component step of its recycling.

Background: Human Rad51 protein (HsRad51) is a homologue of Escherichia coli RecA protein, and involved in homologous recombination. These eukaryotic and bacterial proteins catalyse strand exchange between two homologous DNA molecules, each forming a complex with single‐stranded DNA (ssDNA) and ATP as the initial step. Both proteins hydrolyse ATP; however, the role of ATP hydrolysis appears to vary between the two proteins.Results: Measurements using the fluorescence ssDNA analogue, poly(1,N6‐etheno‐deoxyadenosine), indicate that ATP affects the HsRad51–ssDNA complex, promoting two conformational states: one transient, rather rigid transition state and a final more flexible state. While ADP lowers the affinity of RecA protein to ssDNA, it is found to rather stabilize the HsRad51–ssDNA complex. ADP does not activate the strand exchange by HsRad51 but instead stimulates annealing between complementary ssDNAs.Conclusions: The hydrolysis of ATP promotes a transition of the HsRad51–ssDNA...

Cellular homeostasis is governed by removal of damaged organelles and protein aggregates by selective autophagy mediated by cargo adaptors such as p62/SQSTM1. Autophagosomes can assemble in specialized cup-shaped regions of the endoplasmic reticulum (ER) known as omegasomes, which are characterized by the presence of the ER protein DFCP1/ZFYVE1. The function of DFCP1 is unknown, as are the mechanisms of omegasome formation and constriction. Here, we demonstrate that DFCP1 is an ATPase that dimerizes in an ATP-dependent fashion. Whereas depletion of DFCP1 had a minor effect on bulk autophagic ux, DFCP1 was required to maintain the autophagic ux of p62 under both fed and starved conditions, and this was dependent on its ability to bind and hydrolyse ATP. While DFCP1 mutants defective in ATP binding or hydrolysis localized to forming omegasomes, these omegasomes failed to constrict. Consequently, the release of nascent autophagosomes from omegasomes was markedly delayed. DFCP1 was found to associate with ubiquitinated proteins, and degradation of ubiquitinated cargoes such as protein aggregates, mitochondria and micronuclei was strongly inhibited when DFCP1 was knocked out or mutated. Thus, DFCP1 mediates ATPase-driven constriction of omegasomes to release autophagosomes for selective autophagy.