Cardiolipin activation of dnaA protein, the initiation protein of replication in Escherichia coli

ATP binding to dnaA protein is essential for its action in initiating the replication of plasmids that bear the unique origin of the Escherichia coli chromosome (oriC). ADP bound to that site renders dnaA protein inactive for replication. Diphosphatidylglycerol (cardiolipin), a diacidic membrane phospholipid, displaces the bound nucleotide, and in the presence of components that reconstitute replication, fully reactivates the inert ADP form of dnaA protein. The monacidic phosphatidylglycerol is one-tenth as active as cardiolipin, whereas the neutral phosphatidylethanolamine, the principal E. coli phospholipid, is inactive. Fluphenazine, a tranquilizer drug, blocks cardiolipin activation of dnaA protein, in keeping with the inhibitory action of such agents on phospholipid-dependent enzymes. With the use of this drug to terminate cardiolipin action, dependence of the activation on time, elevated temperature, and high levels of ATP was demonstrated. Cardiolipin binding of nucleotide-free dnaA protein prevents binding of ATP and initiation of oriC replication. Removal of a fatty acid from cardiolipin by phospholipase A reverses this inhibitory effect. The strong and specific interaction of cardiolipin, a cell membrane component, with an essential nucleotide-binding site of dnaA protein, the protein essential for the initiation of chromosome replication, may be an important element in regulating the cell cycle.     

Negative control of DNA replication by hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli.

DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by ATP. ATP bound to DnaA protein is slowly hydrolyzed to ADP, but the physiological role of ATP hydrolysis is unclear. We constructed, by site-directed mutagenesis, mutated DnaA protein with lower ATPase activity, and we examined its function in vitro and in vivo. The ATPase activity of purified mutated DnaA protein (Glu204-->Gln) decreased to one-third that of the wild-type DnaA protein. The mutation did not significantly affect the affinity of DnaA protein for ATP or ADP. The mutant dnaA gene showed lethality in wild-type cells but not in cells growing independently of the function of oriC. Induction of the mutated DnaA protein in wild-type cells caused an overinitiation of DNA replication. Our results lead to the thesis that the intrinsic ATPase activity of DnaA protein negatively regulates chromosomal DNA replication in E. coli cells.     

Fate of the DnaA initiator protein in replication at the origin of the Escherichia coli chromosome in vitro

The dnaA initiator protein binds specific sequences in the 245-base pair Escherichia coli origin (oriC) to form a series of complexes which eventually are opened enough to admit dnaB helicase into a prepriming complex (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755). ATP bound to a high-affinity site on dnaA protein is the preferred form for one or more of the early stages, but an elevated level of ATP is needed for a later stage; further evidence for a low-affinity site has now been obtained. We find that at limiting levels of dnaA protein only the ATP form produces an active initial complex; neither the ADP nor the non-nucleotide forms are effective. Augmentation of the activity of a limiting level of the ATP form of dnaA protein by the otherwise inert ADP form implies that at some stage of initiation both forms are active. The dnaA protein is essential up to the stage of forming the prepriming complex; upon salt dissociation from an oriC complex, the protein can be recycled to function at a fresh origin. Distinctive conformational states of the ATP form are implied by interactions with oriC DNA, by the influence of phospholipids on accelerating nucleotide exchange, and by the susceptibility to proteolytic cleavage.     

The role of tightly bound ADP on chloroplast ATPase

Isolated chloroplast coupling factor 1 ATPase is known to retain about 1 mol of tightly bound ADP/mol of enzyme. Some experimental results have given evidence that the bound ADP is at catalytic sites, but this view has not been supported by observations of a slow replacement of the bound ADP when CaATP or MgATP is added. The experiments reported in this paper show why a slow replacement of ADP bound at a catalytic site can occur. When coupling factor 1, labeled with tightly bound [3H]ADP, is exposed to Mg2+ or Ca2+ prior to the addition of MgATP or CaATP, a pronounced lag in the onset of ATP hydrolysis is observed, and only slow replacement of the [3H]ADP occurs. Mg2+ or Ca2+ can induce inhibition very rapidly, as if an inhibited form of the enzyme results whenever the enzyme with tightly bound ADP encounters Mg2+ or Ca2+ prior to ATP. The inhibited form can be slowly reactivated by incubation with EDTA, although some irreversible loss in activity is encountered. In contrast, when MgATP or CaATP is added to enzyme depleted of Mg2+ and Ca2+ by incubation with EDTA, a rapid onset of ATP hydrolysis occurs and most of the tightly bound [3H]ADP is released within a few seconds, as expected for binding at a catalytic site. The Mg2+-induced inhibition of both the ATPase activity and the lack of replacement of tightly bound [3H] ADP can be largely prevented by incubation with Pi under conditions favoring Pi addition to the site containing the tightly bound ADP. Our and other results can be explained if enzyme catalysis is greatly hindered when MgADP or CaADP without accompanying Pi is tightly bound at one of the three catalytic sites on the enzyme in a high affinity conformation.     

Phospholipids promote dissociation of ADP from the Mycobacterium avium DnaA protein

The biochemical aspects of the initiation of DNA replication in Mycobacterium avium are unknown. As a first step towards understanding this process, M. avium DnaA protein, the counterpart of Escherichia coli replication initiator protein, was overproduced in E. coli with an N-terminal histidine tag and purified to homogeneity on a nickel affinity column. The recombinant DnaA protein bound both ATP and ADP with high affinity and showed a weak ATPase activity. ADP, following the hydrolysis of ATP, remained bound to the protein strongly and the exchange of ATP for bound ADP was found to be weak. Acidic phospholipids such as phosphatidylinositol, phosphatidylglycerol, and cardiolipin, promoted the dissociation of ADP from the DnaA protein, whereas the neutral phospholipid, phosphatidylethanolamine, did not. The phospholipid promoted dissociation of ADP from DnaA protein was stimulated in the presence of the M. avium origin of replication. We suggest that the initiation of DNA replication in M. avium involves an interplay among DnaA, adenine nucleotides and phospholipids.     

Purification and characterization of an initiation protein for chromosomal replication, DnaA, in Bacillus subtilis

Bacillus subtilis DnaA protein was overproduced by a recombinant plasmid containing B. subtilis dnaA gene in a mutant Escherichia coli strain which is deficient in its own DnaA and RNaseH. The protein was purified to near homogeneity as judged by SDS-PAGE analysis. The purified protein binds preferentially to DNA fragments which are derived from flanking regions of the B. subtilis dnaA gene and contain various numbers of the repeat of 9 nucleotides, TTATCCACA, and closely related sequences. The purified protein binds ATP with high affinity (Kd = 0.02 microM) and ADP with less affinity, but does not bind cAMP. ATP stimulates the binding of the DnaA protein to the repeated sequences. DNaseI footprinting experiments demonstrated that the DnaA bound first to the consensus 9-mer and then to sequences differing by one base from the consensus. Sequences differing by two bases from the consensus were bound by the DnaA only when they were located contiguous to the strong DnaA-boxes. The three DnaA-box clusters, incA, incB, and incC, derived from the replication origin region of the B. subtilis chromosome showed different levels of growth inhibition when they were introduced into B. subtilis. We demonstrated by assaying competition for DnaA-binding among the DnaA-box clusters that there is a good correlation between the degree of growth inhibition by DnaA-box clusters in vivo and their strength of binding to the DNaA protein in vitro.     

Chymotryptic cleavage of alpha-subunit in E1-forms of renal (Na+ + K+)-ATPase: effects on enzymatic properties, ligand binding and cation exchange

Chymotrypsin in NaCl medium at low ionic strength rapidly cleaves a bond in the N-terminal half of the alpha-subunit of pure membrane-bound (Na+ + K+)-ATPase from outer renal medulla. Secondary cleavage is very slow and the alpha-subunit can be converted almost quantitatively to a 78 kDa fragment. The sensitive bond is exposed to cleavage when the protein is stabilized in the E1 form by binding of Na+ or nucleotides. The bond is protected in medium containing KCl (E2K form), but it is exposed when ADP or ATP are added (E1KATP form). Fluorescence analysis and examination of ligand binding and enzymatic properties of the cleaved protein demonstrate that cleavage of the bond stabilizes the protein in the E1 form with sites for tight binding of nucleotides and cations exposed to the medium. About two 86Rb ions are bound per cleaved alpha-subunit with normal affinity (Kd = 9 microM). The bound Rb+ is not displaced by ATP or ADP. The nucleotide-potassium antagonism is abolished and ATP is bound with high affinity both in NaCl and in KCl media. Na+-dependent phosphorylation is quantitatively recovered in the 78 kDa fragment, but the affinity for binding of [48V]vanadate is very low after cleavage. ADP-ATP exchange is stimulated 4-5-fold by cleavage; while nucleotide dependent Na+-Na+, K+-K+, or Na+-K+ exchange are abolished. Cleavage with chymotrypsin in NaCl at the N-terminal side of the phosphorylated residue thus stabilizes the E1 form of the protein and abolishes cation exchange and conformational transitions in the protein although binding of cations, nucleotides and phosphate is preserved. In contrast, cleavage with trypsin in KCl at the C-terminal side of the phosphorylated residue does not interfere with E1-E2 transitions and Na+-Na+ or K+-K+ exchange. This data support the notion that cation exchange and E1-E2 transitions are thightly coupled.     

The dnaA initiator protein binds separate domains in the replication origin of Escherichia coli

After binding to its four 9-mer boxes in the 245-base pair Escherichia coli replication origin (oriC), dnaA protein effects the formation of an "open complex" in an adjacent region made up of three 13-mers (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755). This open complex formation requires the ATP form of dnaA protein assisted by HU protein (Sekimizu, K., Bramhill, D., and Kornberg, A. (1987) Cell 50, 259-265). We now provide direct evidence that dnaA protein binds the 13-mers, sequences that bear no resemblance to the 9-mer box. The evidence is (i) displacement of dnaA protein from the open complex by oriC or by a synthetic oligonucleotide containing the 13-mers, but not by a mutant of oriC lacking the 13-mers; (ii) filter binding of the synthetic (13-mer) oligonucleotide by dnaA protein; and (iii) requirement for the ATP form of dnaA protein assisted by HU protein for temperature-dependent binding to the 13-mer region. Controlled proteolysis of dnaA protein results in a prompt loss of oriC binding; an NH2-terminal 30-kDa peptide contains the domain that binds ATP and phospholipids known to destabilize the tightly bound ATP.     

The intrinsic ATPase activity of Mycobacterium tuberculosis DnaA promotes rapid oligomerization of DnaA on oriC

Oligomerization of the initiator protein, DnaA, on the origin of replication (oriC) is crucial for initiation of DNA replication. Studies in Escherichia coli (Gram-negative) have revealed that binding of DnaA to ATP, but not hydrolysis of ATP, is sufficient to promote DnaA binding, oligomerization and DNA strand separation. To begin understanding the initial events involved in the initiation of DNA replication in Mycobacterium tuberculosis (Gram-positive), we investigated interactions of M. tuberculosis DnaA (DnaA(TB)) with oriC using surface plasmon resonance in the presence of ATP and ADP. We provide evidence that, in contrast to what is observed in E. coli, ATPase activity of DnaA(TB) promoted rapid oligomerization on oriC. In support, we found that a recombinant mutant DnaA(TB) proficient in binding to ATP, but deficient in ATPase activity, did not oligomerize as rapidly. The corresponding mutation in the dnaA gene of M. tuberculosis resulted in non-viability, presumably due to a defect in oriC-DnaA interactions. Dimethy sulphate (DMS) footprinting experiments revealed that DnaA(TB) bound to DnaA boxes similarly with ATP or ADP. DnaA(TB) binding to individual DnaA boxes revealed that rapid oligomerization on oriC is triggered only after the initial interaction of DnaA with individual DnaA boxes. We propose that ATPase activity enables the DnaA protomers on oriC to rapidly form oligomeric complexes competent for replication initiation.     

The effects of octylglucoside on the interactions of chloroplast coupling factor 1 (CF1) with adenine nucleotides

The effects of octylglucoside (OcGlc) micelles, which stimulate a Mg-specific ATPase activity in chloroplast coupling factor 1 [Pick, U. and Bassilian, S. (1982) Biochemistry, 21, 6144-6152], on the interactions of the enzyme with adenine nucleotides have been studied. 1. OcGlc specifically accelerates the binding and the release of ADP but not of ATP or adenosine 5'[beta, gamma-imido]triphosphate (AdoPP[NH]P) from the tight-sites. The binding affinity for ADP and for ATP is only slightly decreased (twofold) by the detergent. ATP competitively inhibits the binding of ADP and vice versa in the presence or absence of OcGlc. 2.OcGlc-induced inactivation of CF1-ATPase is correlated with the release of bound nucleotides. In the absence of medium nucleotides ADP X CF1 is rapidly inactivated while ATP X CF1 and AdoPP[NH]P X CF1 are slowly inactivated by OcGlc in parallel with the release of bound nucleotide. In contrast, low concentrations of either ATP or ADP in the medium effectively protect against OcGlc inactivation while AdoPP[NH]P, whose binding to CF1 is inhibited by OcGlc, is ineffective even at millimolar concentrations. The results suggest that the occupancy of the tight-sites protects the enzyme against OcGlc-induced inactivation. 3. Mg ions specifically inhibit the release of bound ADP and the OcGlc-induced inactivation of CF1. High concentrations of medium ATP and ADP (K50 = 100 microM) also inhibit the OcGlc-induced release of bound nucleotides in an EDTA medium. In contrast, in the absence of OcGlc, medium ADP and ATP accelerate the release of bound adenine nucleotides. 4. Mg-ATP in the presence of OcGlc stimulates the release of bound ADP from CF1. Bound ATP is neither released nor hydrolyzed at the tight-sites under these conditions where medium ATP is rapidly hydrolyzed. Mg-ADP stimulates the release of bound ADP only in the presence of inorganic phosphate or of phosphate analogs, e.g. arsenate, pyrophosphate or selenate. 5. It is suggested that: (a) ATP and ADP bind to the same tight-sites, but OcGlc activation specifically accelerates the exchange of bound ADP at the site. (b) CF1 contains low affinity adenine nucleotide binding sites which may be the catalytical sites and which influence the tight-sites by cooperative interactions. (c) Mg-ATP in the presence of OcGlc induces a conformational change at the catalytical site which accelerates the release of ADP from the tight-site. The implications of these results to the role of adenine nucleotides in the regulation and mechanism of ATP hydrolysis by CF1 are discussed.     

Differences in G-actin containing bound ATP or ADP: the Mg2+-induced conformational change requires ATP

The role of adenosine 5'-triphosphate (ATP) in the Mg2+-induced conformational change of rabbit skeletal muscle G-actin has been investigated by comparing actin containing bound ADP with actin containing bound ATP. As previously described [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886], N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled G-actin containing ATP undergoes a time-dependent Mg2+-induced fluorescence change that reflects a conformational change in the actin. Addition of Mg2+ to labeled G-actin containing ADP gives no fluorescence change, suggesting that the conformational change does not occur. The fluorescence change can be restored on the addition of ATP. Examination of the time courses of these experiments suggests that ATP must replace ADP prior to the Mg2+-induced change. The Mg2+-induced polymerization of actin containing ADP is extraordinarily slow compared to that of actin containing ATP. The lack of the Mg2+-induced conformational change, which is an essential step in the Mg2+-induced polymerization, is probably the cause for the very slow polymerization of actin containing ADP. On the other hand, at 20 degrees C, at pH 8, and in 2 mM Mg2+, the elongation rate from the slow growing end of an actin filament, measured by using the protein brevin to block growth at the fast growing end, is only 4 times slower for actin containing ADP than for actin containing ATP.     

Binding of nucleotides to the ATP-dependent protease La from Escherichia coli

A critical enzyme in protein breakdown in Escherichia coli is the ATP-hydrolyzing protease La, the lon gene product. In order to clarify the role of ATP in proteolysis, we studied ATP and ADP binding to this enzyme using rapid gel filtration to separate free from bound ligands. In the presence of Mg2+ or Mn2+ and 10 microM ATP, two molecules of ATP were bound to the tetrameric enzyme, while at 100 microM ATP (or higher), four ATP molecules were bound, both at 0 and 37 degrees C. Protease La thus has two high affinity sites (S0.5 less than 10(-7) M) for ATP and two lower affinity sites (S0.5 = 12-15 microM). Binding was reversible. In the absence of a divalent ion, ATP bound to only two sites. However, much lower Mg2+ concentrations (50 microM) were required for maximal ATPase binding than for maximal proteolytic and ATPase activity (2 mM). Decavanadate, which is a potent inhibitor of proteolysis, also blocked ATP binding, but orthovanadate had neither effect. Different ATP analogs bind to these sites in distinct ways. Adenyl-5'-yl imidodiphosphate binds to only one high affinity site, while adenyl-5'-yl methylene monophosphonate binds to two. Nevertheless, both non-metabolizable analogs can activate oligopeptide hydrolysis as well as ATP. Although binding of a single nucleotide can activate peptide hydrolysis, occupancy of all four sites appears necessary for maximal protein breakdown. The ATP molecules on all four sites are hydrolyzed rapidly. The Pi is released, but ADP remains on the enzyme. ADP binds to the same four sites, but this process does not require divalent ions. Protease La shows higher affinity for ADP than for ATP. Therefore, in vivo, ADP should inhibit ATP binding and protease La function.     

The chromosome origin of Escherichia coli stabilizes DnaA protein during rejuvenation by phospholipids.

DnaA protein (the initiator protein) binds and clusters at the four DnaA boxes of the Escherichia coli chromosomal origin (oriC) to promote the strand opening for DNA replication. DnaA protein activity depends on the tight binding of ATP; the ADP form of DnaA protein, generated by hydrolysis of the bound ATP, is inactive. Rejuvenation of ADP-DnaA protein, by replacement with ATP, is catalyzed by acidic phospholipids in a highly fluid bilayer. We find that interaction of DnaA protein with oriC DNA is needed to stabilize DnaA protein during this rejuvenation process. Whereas DnaA protein bound to oriC DNA responds to phospholipids, free DnaA protein is inactivated by phospholipids and then fails to bind oriC. Furthermore, oriC DNA facilitates the high affinity binding of ATP to DnaA protein during treatment with phospholipids. A significant portion of the DnaA protein associated with oriC DNA can be replaced by the ADP form of the protein, suggesting that all of the DnaA protein bound to oriC DNA need not be rejuvenated between rounds of replication.     

Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.     

Interaction of ADP and ATP with noncatalytic sites of isolated and membrane-bound chloroplast coupling factor CF<Subscript>1</Subscript>

This study of ATP and ADP binding to noncatalytic sites of membrane-bound CF1 (ATP synthase) revealed two noncatalytic sites with different specificities and affinities for nucleotides. One of these is characterized by a high affinity and specificity to ADP (Kd=2.6+/-0.3 microM). However, a certain increase in ADP apparent dissociation constant at high ATP/ADP ratio in the medium allows a possibility that ATP binds to this site as well. The other site displays high specificity to ATP. When the ADP-binding site is vacant, it shows a comparatively low affinity for ATP, which greatly increases with increasing ADP concentration accompanied by filling of the ADP-binding site. The reported specificities of these two sites are independent of thylakoid membrane energization, since both in the dark and in the light the ratios of ATP/ADP tightly bound to the noncatalytic sites were very close. The difference in noncatalytic site affinity for ATP and ADP is shown to depend on the amount of delta subunit in a particular sample. Thylakoid membrane ATP synthase, with stoichiometric content of delta-subunit (one delta-subunit per CF1 molecule), showed the maximal difference in ADP and ATP affinities for the noncatalytic sites. For CF1, with substoichiometric delta subunit values, this difference was less, and after delta subunit removal it decreased still more.     

Inactivation of Escherichia coli DnaA protein by DNA polymerase III and negative regulations for initiation of chromosomal replication

Genetic and biochemical evidence indicates that initiation of chromosomal replication in Escherichia coli occurs in a nucleoprotein complex at the replication origin (oriC) formed with DnaA protein. The frequency of initiation at oriC is tightly regulated to only once per chromosome per cell cycle. To prevent untimely, extra initiations, negative control for initiation is indispensable. Recently, we found that the function of the initiator protein, DnaA, is controlled by DNA polymerase III holoenzyme, the replicase of the chromosome. The ATP-bound form of DnaA protein, an active form for initiation, is efficiently converted to the ADP bound form, an inactive form, since a subunit of the polymerase loaded on DNA (beta subunit sliding clamp) stimulates hydrolysis of ATP bound to DnaA protein. Comparison of this system, RIDA (regulatory inactivation of DnaA), with other systems for negative regulation of initiation is included in this review, and the roles of these systems for concerted control for initiation during the cell cycle are discussed.     

Characteristics of the formation of enzyme-bound ATP from medium inorganic phosphate by mitochondrial F1 adenosinetriphosphatase in the presence of dimethyl sulfoxide

Addition of dimethyl sulfoxide promotes the formation of enzyme-bound ATP from medium Pi by mitochondrial F1 adenosinetriphosphatase that has tightly bound ADP present. Measurements are reported of medium Pi in equilibrium H18OH exchange and of the dependence of formation of enzyme-bound ATP on Pi concentration. Attainment of an apparent equilibrium between medium Pi and bound ATP requires longer than 30 min, even though the rates of Pi binding and release after apparent equilibrium is reached would suffice for a faster approach to equilibrium. Slow protein conformational changes or other unknown modulating factors may be responsible for the slow rate of bound ATP formation. After apparent equilibrium is reached, each Pi that binds to the enzyme reversibly forms ATP about 50 times before being released to the medium. The rate of interconversion of bound ATP to bound ADP and Pi is much slower than that in the absence of dimethyl sulfoxide as measured with sufficiently low ATP concentrations so that single-site catalysis is favored. Although the interconversion rate is slowed, the equilibrium constant for bound ATP formation from bound ADP and Pi is not far from unity. Dimethyl sulfoxide favors the formation of enzyme-bound ATP by promoting the competent binding of Pi to enzyme with ADP bound at a catalytic site rather than by promoting formation of bound ATP from bound ADP and Pi.     

Independent ATPase activity of Hsp90 subunits creates a flexible assembly platform

The ATPase activity of the molecular chaperone Hsp90 is essential for its function in the assembly of client proteins. To understand the mechanism of human Hsp90, we have carried out a detailed kinetic analysis of ATP binding, hydrolysis and product release. ATP binds rapidly in a two-step process involving the formation of a diffusion-collision complex followed by a conformational change. The rate-determining step was shown to be ATP hydrolysis and not subsequent ADP dissociation. There was no evidence from any of the biophysical measurements for cooperativity in either nucleotide binding or hydrolysis for the dimeric protein. A monomeric fragment, lacking the C-terminal dimerisation domain, showed no dependence on protein concentration and, therefore, subunit association for activity. The thermodynamic linkage between client protein binding and nucleotide affinity revealed ATP bound Hsp90 has a higher affinity for client proteins than the ADP bound form. The kinetics are consistent with independent Michaelis-Menten catalysis in each subunit of the Hsp90 dimer. We propose that Hsp90 functions in an open-ring configuration for client protein activation.     

Discrimination of ATP,ADP, and AMPPNP by chaperonin GroEL: hexokinase treatment revealed the exclusive role of ATP

The double ring chaperonin GroEL binds unfolded protein, ATP, and GroES to the same ring, generating the cis ternary complex in which folding occurs within the cavity capped by GroES (cis folding). The functional role of ATP, however, remains unclear since several reports have indicated that ADP and AMPPNP (5'-adenylyl-beta,gamma-imidodiphosphate) are also able to support the formation of the cis ternary complex and the cis folding. To minimize the effect of contaminated ATP and adenylate kinase, we have included hexokinase plus glucose in the reaction mixtures and obtained new results. In ADP and AMPPNP, GroES bound quickly to GroEL but bound very slowly to the GroEL loaded with unfolded rhodanese or malate dehydrogenase. ADP was unable to support the formation of cis ternary complex and cis folding. AMPPNP supported cis folding of malate dehydrogenase to some extent but not cis folding of rhodanese. In the absence of hexokinase, apparent cis folding of rhodanese and malate dehydrogenase was observed in ADP and AMPPNP. Thus, the exclusive role of ATP in generation of the cis ternary complex is now evident.     

Characterization of Escherichia coli DnaAcos protein in replication systems reconstituted with highly purified proteins

Excessive initiation of chromosomal replication occurs in the dnaAcos mutant at 30°C. Whereas purified wild-type DnaA protein binds ATP and ADP tightly, DnaAcos protein is defective for such nucleotide binding. As initiation is a multistep reaction and DnaA protein functions at each step, activities of DnaAcos protein need to be examined precisely. DnaAcos protein specifically bound a DNA fragment containing the chromosomal replication origin with an affinity similar to that seen with the wild-type protein. In a system reconstituted with purified proteins at 30°C, the mutant protein initiated replication of single-stranded DNA that contains a DnaA-binding hairpin structure. Thus, DnaAcos protein basically sustains affinity to a DnaA-binding sequence and functions in the loading of DnaB helicase onto single-stranded DNA. Thermal stabilities of wild-type DnaA and DnaAcos activities were comparable. Unlike wild-type DnaA protein, DnaAcos protein was inactive for minichromosomal replication in systems reconstituted with purified proteins in which the ATP-bound form of DnaA protein is required for initiation. Taken together, the data indicate that the prominent defect in DnaAcos protein appears to be the inability to bind nucleotide.