The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE

Molecular chaperones of the Hsp70 class bind unfolded polypeptide chains and are thought to be involved in the cellular folding pathway of many proteins. DnaK, the Hsp70 protein of Escherichia coli, is regulated by the chaperone protein DnaJ and the cofactor GrpE. To gain a biologically relevant understanding of the mechanism of Hsp70 action, we have analyzed a model reaction in which DnaK, DnaJ, and GrpE mediate the folding of denatured firefly luciferase. The binding and release of substrate protein for folding involves the following ATP hydrolysis-dependent cycle: (i) unfolded luciferase binds initially to DnaJ; (ii) upon interaction with luciferase-DnaJ, DnaK hydrolyzes its bound ATP, resulting in the formation of a stable luciferase-DnaK-DnaJ complex; (iii) GrpE releases ADP from DnaK; and (iv) ATP binding to DnaK triggers the release of substrate protein, thus completing the reaction cycle. A single cycle of binding and release leads to folding of only a fraction of luciferase molecules. Several rounds of ATP-dependent interaction with DnaK and DnaJ are required for fully efficient folding.     

The rpoD gene functions as a multicopy suppressor for mutations in the chaperones, CbpA, DnaJ and DnaK, in Escherichia coli

The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli. The dnaJ- cbpA- double-null mutant exhibits severe defects in cell growth, namely, a very narrow temperature range for growth. To gain insight into the functions of CbpA as well as DnaJ, we isolated a multicopy suppressor gene that permits this dnaJ- cbpA- mutant to grow normally at low temperatures. The suppressor gene was identified as rpoD, the gene that encodes the major sigma 70. The biological implications of this finding are examined and discussed.     

Chaperone protein GrpE and the GroEL/GroES complex promote the correct folding of tobacco mosaic virus coat protein for ribonucleocapsid assembly in vivo

Several prokaryotic chaperone proteins were shown to promote the correct folding and in vivo assembly of tobacco mosaic virus coat protein (TMV CP) using a chimaeric RNA packaging system in control or chaperone-deficient mutant strains of Escherichia coli. Mutations in groEL or dnaK reduced the amount of both total and soluble TMV CP, and the yield of assembled TMV-like particles, several-fold. Thus both GroEL and DnaK have significant direct or indirect effects on the overall expression, stability, folding and assembly of TMV CP in vivo. In contrast, while cells carrying a mutation in grpE expressed TMV CP to a higher overall level than control E. coli, the amounts of both soluble CP and assembled TMV-like particles were below control levels, suggesting a negative effect of GrpE on overall CP accumulation, but positive role(s) in CP folding and assembly. Curiously, cells with mutations in groES and, to a lesser extent, dnaJ expressed total, soluble and assembled forms of TMV CP significantly above control values, suggesting some form of negative control by these chaperone proteins. To avoid pleiotropic effects or artefacts in chaperone-null mutants, selected chaperone proteins were also over-expressed in control E. coli cells. Overproduction of GroEL or GroES alone had little effect. However, co-overexpression of GroEL and GroES resulted in a two-fold increase in soluble TMV CP and a four-fold rise in assembled TMV-like (pseudovirus) particles in vivo. Moreover, TMV CP was shown to interact directly with GroEL in vivo. Together, these results suggest that GrpE and the GroEL/GroES chaperone complex promote the correct folding and assembly of TMV CP into ribonucleocapsids in vivo.     

The mediator for stringent control, ppGpp, binds to the beta-subunit of Escherichia coli RNA polymerase

OBJECTIVE: Evaluate the behavioral reputation and peer acceptance of children diagnosed and treated for brain tumors. METHOD: Twenty-eight children surviving brain tumors (8-18 years of age) were compared to 28 nonchronically ill, same classroom, same gender comparison peers (COMP). Peer, teacher, and self-report data were collected. RESULTS: Relative to COMP, children who had been diagnosed with brain tumors received fewer friendship nominations from clasmates and were described by peer, teacher, and self-report as socially isolated. Although they were no longer receiving therapy for their disease, peers perceived brain tumor survivors as being sick, more fatigued, and often absent from school. CONCLUSIONS: These data suggest that children surviving brain tumors are at risk for social difficulties even after treatment ends, although the specific cause(s) for this vulnerability were not investigated in the current study.     

Kinetic analysis of the RNA-dependent adenosinetriphosphatase activity of DbpA, an Escherichia coli DEAD protein specific for 23S ribosomal RNA

The adenosinetriphosphatase (ATPase) activity of the Escherichia coli DEAD protein DbpA is unusual in that it is specifically stimulated by 23S ribosomal RNA (rRNA). A coupled spectroscopic ATPase assay was used to investigate the interaction of DbpA with RNA and ATP. A 153-base fragment of domain V of 23S rRNA is kinetically identical to intact, native rRNA in activating DbpA: kcat = 600 min-1, Kapp(RNA) = 10 nM, and Km(ATP) = 120 microM. The ATPase turnover in the absence of RNA is 0.25 min-1. Fragments of 23S rRNA lacking this site (nucleotides 2454-2606) are essentially inactive, as are other RNAs such as poly(A) and tRNA. The relative RNA specificity of DbpA ranges from 10(3) to 10(6) [kmax/Kapp(RNA)]. The interaction with this small RNA fragment was further investigated with regard to stoichiometry, pH, salt and temperature. DbpA is not activated by E. coli ribosomes, nor by large subunits, while denatured ribosomes stimulate full ATPase activity. Taken together with the tight, site-specific binding to naked, unmodified 23S rRNA, this suggests a role for DbpA in ribosome biogenesis rather than translation.     

Architecture of a complex between the sigma70 subunit of Escherichia coli RNA polymerase and the nontemplate strand oligonucleotide. Luminescence resonance energy transfer study

We used luminescence energy transfer measurements to determine the localization of 5'- and 3'-ends of a 12-nucleotide nontemplate strand oligonucleotide bound to sigma70 holoenzyme. Five single reactive cysteine mutants of sigma70 (cysteine residues at positions 1, 59, 366, 442, and 596) were labeled with a europium chelate fluorochrome (donor). The oligonucleotide was modified at the 5'- or at the 3'-end with Cy5 fluorochrome (acceptor). The energy transfer was observed upon complex formation between the donor-labeled sigma70 holoenzyme and the acceptor-labeled nontemplate strand oligonucleotide, whereas no interaction was observed with the template strand oligonucleotide. The oligonucleotide was bound in one preferred orientation. This observation together with the sequence specificity of single-stranded oligonucleotide interaction suggests that two mechanisms of discrimination between the template and nontemplate strand are used by sigma70: sequence specificity and strand polarity specificity. The bound oligonucleotide was found to be close to residue 442, confirming that the single-stranded DNA binding site of sigma70 is located in an alpha-helix containing residue 442. The 5'-end of the oligonucleotide was oriented toward the COOH terminus of the helix.     

Affinity of the periplasmic chaperone Skp of Escherichia coli for phospholipids, lipopolysaccharides and non-native outer membrane proteins. Role of Skp in the biogenesis of outer membrane protein

The Skp protein of Escherichia coli has been proposed to be a periplasmic molecular chaperone involved in the biogenesis of outer membrane proteins. In this study, evidence is obtained that Skp exists in two different states characterized by their different sensitivity to proteases. The conversion between these states can be modulated in vitro by phospholipids, lipopolysaccharides and bivalent cations. Skp is able to associate with and insert into phospholipid membranes in vitro, indicating that it may associate with phospholipids in the inner and/or outer membrane in vivo. In addition, it interacts specifically with outer membrane proteins that are in their non-native state. We propose that Skp is required in vivo for the efficient targeting of unfolded outer membrane proteins to the membrane.     

Diffuse adherence, ST-I enterotoxin and CFA/IV colonization factor are encoded by the same plasmid in the Escherichia coli O29:H21 strain

Escherichia coli O29:H21 is a human enterotoxigenic serotype that produces heat-stable (ST-I) enterotoxin, adheres diffusely to HeLa cells, and presents colonization factor antigen IV (CFA/IV) composed of CS5CS6 surface antigens. In one strain studied the genes for diffuse adherence and CFA/IV (CS5CS6) production were found to be present in the same plasmid encoding ST-I. The virulence plasmid (Ent) presented two unrelated basic replicons homologous to repFIC and repW. Gene(s) encoding diffuse adherence did not share homology with the probe for F1845 fimbrial adhesin which is responsible for this phenotype in other E. coli strains. Ent plasmids containing genes for diffuse adherence have not been described previously.     

The effect of acylated polyamine derivatives on polyamine uptake mechanism, cell growth, and polyamine pools in Escherichia coli, and the pursuit of structure/activity relationships

Two acetyl analogues of spermidine and five analogues of spermine were used to determine the structural specificity of the polyamine transport system in Escherichia coli by measuring their ability to compete with [14C]putrescine or [14C]spermine for uptake, as well as to inhibit cell growth, and, finally, to affect the intracellular polyamine pools. Spermine uptake follows simple Michaelis-Menten kinetics (Kt = 24.58 +/- 2.24 microM). In contrast, the putrescine uptake system involves two saturable Michaelis-Menten carriers exhibiting different affinity towards putrescine (Kt = 3.63 +/- 0.43 microM, Kt' = 0.61 +/- 0.10 microM). From the Ki values, it is inferred that N1-5-amino-2-nitrobenzoylspermine is the most effective competitive inhibitor followed by N1-acetylspermine, and then N1,N12-diacetylspermine. N1-acetylspermidine and N8-acetylspermidine also inhibit competitively the uptake of spermine, the latter being the most effective inhibitor. In addition, the above-mentioned analogues inhibit identically one of the carriers of putrescine uptake, suggesting the existence of a common transporter for both putrescine and spermine. The order of analogue potency, regarding the other carrier of putrescine is as follows: N1,N12-diacetylspermine approximately N1-5-amino-2-nitro-benzoylspermine > N1-acetylspermine. Both N1-acetylspermidine (Ki = 753 +/- 25 microM, Ki' = 128 +/- 5 microM) and N8-acetylspermidine (Ki = 22.4 +/- 0.4 microM, Ki' = 279 +/- 3 microM) also cause competitive inhibition of putrescine uptake, however with inverse affinity towards the putrescine carriers. Neither N4,N9-diacetylspermine, nor N1,N4-bis(beta-alanyl)diaminobutane affect the uptake of any polyamine. Interestingly, none of the acetyl analogues of spermine has a measurable effect on cell growth and cellular polyamine pools, although some of them are accumulated in cells. Based on these findings, the relative significance of the primary and secondary amines and of the chain flexibility as determinants of cellular uptake are discussed.     

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.     

The Escherichia coli FOF1 gammaM23K uncoupling mutant has a higher K0.5 for Pi. Transition state analysis of this mutant and others reveals that synthesis and hydrolysis utilize the same kinetic pathway

The Escherichia coli FOF1 ATP synthase uncoupling mutation, gammaM23K, was found to increase the energy of interaction between gamma and beta subunits, prevent the proper utilization of binding energy to drive catalysis, and block the enzyme in a Pi release mode. In this paper, the effects of this mutation on substrate binding in cooperative ATP synthesis are assessed. Activation of ATP synthesis by ADP and Pi was determined for the gammaM23K FOF1. The K0.5 for ADP was not affected, but K0.5 for Pi was approximately 7-fold higher even though the apparent Vmax was close to the wild-type level. Wild-type enzyme had a turnover number of 82 s-1 at pH 7.5 and 30 degrees C. During oxidative phosphorylation, the apparent dissociation constant (KI) for ATP was not affected and was 5-6 mM for both wild-type and gammaM23K enzymes. Thus, the apparent binding affinity for ATP in the presence of DeltamuH+ was lowered by 7 orders of magnitude from the affinity measured at the high-affinity catalytic site. Arrhenius analysis of ATP synthesis for the gammaM23K FOF1 revealed that, like those of ATP hydrolysis, the transition state DeltaH was much more positive and TDeltaS was much less negative, adding up to little change in DeltaG. These results suggested that ATP synthesis is inefficient because of an extra bond between gamma and beta subunits which must be broken to achieve the transition state. Analysis of the transition state structures using isokinetic plots demonstrate that ATP hydrolysis and synthesis utilize the same kinetic pathway. Incorporating this information into a model for rotational catalysis suggests that at saturating substrate concentrations, the rate-limiting step for hydrolysis and synthesis is the rotational power stroke where each of the beta subunits changes conformation and affinity for nucleotide.     

Conservation of the 168 divIB gene in Bacillus subtilis W23 and B. licheniformis, and evidence for homology to ftsQ of Escherichia coli

Recent reports indicate that combined anterior cruciate ligament/medial collateral ligament (ACL/MCL) knee injuries are usually associated with a lateral meniscus tear. In our center, snow skiing is the athletic activity most frequently associated with this double-ligament injury complex. A sports-specific analysis was undertaken to evaluate the hypothesis that the snow skiing ligament injury is different from similar injuries caused by other athletic activities. Of a total of 64 acute arthroscopically confirmed tears of both the MCL and ACL, 23 were caused by snow skiing and 41 by nonskiing activities. There were fewer lateral meniscus tears in skiers (43%) when compared with the nonskiers (88%). Skiers also had fewer medial meniscus tears (13%) than did nonskiers (37%). No medial meniscus tears occurred in the absence of a lateral meniscus tear. Although 78% of the skiers were women, only 12% of the nonskiers were women. Skiers were older (average age 35 years) than the nonskiers (average age 28 years). The right knee was injured almost twice as frequently as the left. These data suggest that the double (ACL/MCL) ligament injury in skiers might be distinctly different from that in nonskiers.     

Luminal transport system for choline+ in relation to the other organic cation transport systems in the rat proximal tubule. Kinetics, specificity: alkyl/arylamines, alkylamines with OH, O, SH, NH2, ROCO, RSCO and H2PO4-groups, methylaminostyryl, rhodamine, acridine, phenanthrene and cyanine compounds

The efflux of [3H] choline+ from the proximal tubular lumen was measured by using the stop-flow microperfusion method. The 2-s efflux of [3H] choline+ follows kinetics with a Michaelis constant, Km = 0.18 mmol x l-1, maximal flux, Jmax = 0.43 pmol x cm-1 x s-1 and a permeability term = 38.0 micron2 small middle dots-1. Replacement of Na+ by N-methyl-D-glucamine+ or Li+, or a change of luminal pH do not alter choline+ efflux. Replacement of Na+ by Cs+ inhibits 2-s choline+ (0. 01 mmol x l-1) efflux by 22% and replacement by K+ inhibits by 49%, indicating that the electrical potential difference across the brush border membrane acts as driving force for choline+ transport. Comparing the apparent luminal inhibitory constant values for choline (app. Ki,l,choline+) with the chemical structure of inhibiting substrates, it was found that the inhibitory potency of amines with high pKa values, i.e. high basicity, and of quaternary ammonium compounds (tetraethyl to tetrahexylammonium) increases with their hydrophobicity in a similar manner as was observed previously against the contraluminal N1-methylnicotinamide (NMeN+) transporter and the luminal H+/organic cation (N-methyl-4-phenylpyridinium) (MPP+) exchanger. Independently of their hydrophobicity, an increase in the inhibitory potency of the homologous series of aminoquinolines against the choline+ transporter was observed with increasing pKa values, i.e. increasing basicity, as was found previously against the two other organic cation transporters. A third parameter influencing the interaction with the choline+ transporter is the presence of two amino groups with high pKa values or one amino group and a permanent positive charge, as is documented with the two-ring aminostyryl and rhodamine compounds, as well as three-ring aminoacridine, aminophenanthrene and cyanine compounds. Thus with the aminostyryl, pyridinium+, rhodamine, phenanthridium+ and cyanine+ dyes app.Ki,l,choline+ values of between 0.01 and 0.07 mmol x l-1 have been found. A fourth parameter influencing the choline+ transporter is the presence of an OH group on the C atom next to that bearing the N atom (as in choline+) or an ester-OCOR group (acetylcholine+, butyrylcholine+) or a thioester-SCOR-group (acetylthiocholine+, butyrylthiocholine+); or an -OP(OH)2(OR) group (glycerylphosphoryl-choline+), resulting in app.Ki,l,choline+ values of 0.3-1.0 mmol x l-1. Thus the substrates for the luminal choline+ transporter have general features in common with the luminal H+/organic cation exchanger and the contraluminal organic cation transporter, i.e. hydrophobicity and basicity. Additional parameters for interaction are an OH (or similar) group positioned a favourable distance from the N atom or a second amino/ammonium group in multi-ring compounds.     

Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities

Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P, 2-kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of beta-D-fructose- 6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru-2,6-P2) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166-2175). We have solved the crystal structure of H256A to a resolution of 2. 4 A by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0. 032 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.