Thermodynamic properties of nucleotide-free EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors of its GTPase activity

The thermal transition of elongation factor EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors was studied by differential scanning calorimetry. The effectors of GTPase activity used were the antibiotic kirromycin and the cations Li(+), Na(+), K(+) and NH(4)(+) in the chloride form. The temperature of thermal denaturation and the cooperativity of the transition of nucleotide-free EF-Tu (EF-Tu(f)) in the presence of kirromycin are comparable with those of the EF-Tu x guanosine-5'-[beta,gamma-imido]triphosphate (GppNHp) form, indicating similar conformational states. Increased concentrations of Na(+) and K(+) stabilized EF-Tu(f) in a manner similar to GppNHp. NH(4)(+) decreased the transition temperature of EF-Tu(f) and Li(+) decreased both the temperature and the calorimetric enthalpy of the thermal transition of EF-Tu(f). In the presence of salts, binding of kirromycin had a stabilizing effect on EF-Tu(f). Correlation between the GTPase activity and thermodynamic characteristics of EF-Tu(f) induced by kirromycin in the absence or presence of the cations is discussed.     

Trigger factor from Thermus thermophilus is a Zn2+-dependent chaperone

The ribosome-associated chaperone trigger factor (TF) of Escherichia coli interacts with a variety of newly synthesized polypeptides to assist their correct folding. Here, we report that the TF of thermophilic eubacterium, Thermus thermophilus, arrested spontaneous folding of green fluorescent protein by forming a 1:1 binary complex. The complex was isolable by gel-filtration but was shown to be dynamic because green fluorescent protein was released by alpha-casein in large excess. Unexpectedly, EDTA completely abolished the folding-arrest activity of TF, and analysis revealed that the TF from our preparation contained approximately 0.5 mol Zn2+/mol TF. The folding-arrest activity of TF that was saturated with Zn2+ (approximately 1 mol/mol TF) was twice as efficient as that of untreated TF. Thus, chaperone activity of thermophilic TF is Zn2+-dependent.     

Predicting the binding affinities of misacylated tRNAs for Thermus thermophilus EF-Tu.GTP

The free energies for the binding of 20 different unmodified Escherichia coli elongator aminoacyl-tRNAs to Thermus thermophilus elongation factor Tu (EF-Tu) were determined. When combined with the binding free energies for the same tRNA bodies misacylated with either valine or phenylalanine determined previously [Asahara, H., and Uhlenbeck, O. C. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 3499-3504], these data permit the calculation of the contribution of each esterified amino acid to the total free energy of binding of the complex. The two data sets can also be used to calculate the free energy of binding of EF-Tu to any misacylated E. coli tRNA, and the values agree well with previously published experimental values. In addition, a survey of active misacylated suppressor tRNAs suggests that a minimal threshold of binding free energy for EF-Tu is required for suppression to occur.     

Opposite roles of domains 2+3 of Escherichia coli EF-Tu and Bacillus stearothermophilus EF-Tu in the regulation of EF-Tu GTPase activity

The effect of noncatalytic domains 2+3 on the intrinsic activity and thermostability of the EF-Tu GTPase center was evaluated in experiments with isolated domains 1 and six chimeric variants of mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) EF-Tus. The isolated catalytic domains 1 of both EF-Tus displayed similar GTPase activities at their optimal temperatures. However, noncatalytic domains 2+3 of the EF-Tus influenced the GTPase activity of domains 1 differently, depending on the domain origin. Ecdomains 2+3 suppressed the GTPase activity of the Ecdomain 1, whereas those of BstEF-Tu stimulated the Bstdomain 1 GTPase. Domain 1 and domains 2+3 of both EF-Tus positively cooperated to heat-stabilize their GTPase centers to attain optimal activity at a temperature close to the optimal growth temperature of either organism. This can be explained by a stabilization effect of domains 2+3 on alpha-helical regions of the G-domain as revealed by CD spectroscopy.     

In vitro trans-translation of Thermus thermophilus: ribosomal protein S1 is not required for the early stage of trans-translation

Transfer-messenger RNA (tmRNA) plays a dual role as a tRNA and an mRNA in trans-translation, during which the ribosome replaces mRNA with tmRNA encoding the tag-peptide. These processes have been suggested to involve several tmRNA-binding proteins, including SmpB and ribosomal protein S1. To investigate the molecular mechanism of trans-translation, we developed in vitro systems using purified ribosome, elongation factors, tmRNA and SmpB from Thermus thermophilus. A stalled ribosome in complex with polyphenylalanyl-tRNA(Phe) was prepared as a target of tmRNA. A peptidyl transfer reaction from polyphenylalanyl-tRNA(Phe) to alanyl-tmRNA was observed in an SmpB-dependent manner. The next peptidyl transfer to aminoacyl-tRNA occurred specifically to the putative resume codon for the tag-peptide, which was confirmed by introducing a mutation in the codon. Thus, the in vitro systems developed in this study are useful to investigate the early steps of trans-translation. Using these in vitro systems, we investigated the function of ribosomal protein S1, which has been believed to play a role in trans-translation. Although T. thermophilus S1 tightly bound to tmRNA, as in the case of Escherichia coli S1, it had little or no effect on the early steps of trans-translation.     

Novel protein and Mg2+ configurations in the Mg2+GDP complex of the SRP GTPase ffh

Ffh is the signal sequence recognition and targeting subunit of the prokaryotic signal recognition particle (SRP). Previous structural studies of the NG GTPase domain of Ffh demonstrated magnesium-dependent and magnesium-independent binding conformations for GDP and GMPPNP that are believed to reflect novel mechanisms for exchange and activation in this member of the GTPase superfamily. The current study of the NG GTPase bound to Mg(2+)GDP reveals two new binding conformations-in the first the magnesium interactions are similar to those seen previously, however, the protein undergoes a conformational change that brings a conserved aspartate into its second coordination sphere. In the second, the protein conformation is similar to that seen previously, but the magnesium coordination sphere is disrupted so that only five oxygen ligands are present. The loss of the coordinating water molecule, at the position that would be occupied by the oxygen of the gamma-phosphate of GTP, is consistent with that position being privileged for exchange during phosphate release. The available structures of the GDP-bound protein provide a series of structural snapshots that illuminate steps along the pathway of GDP release following GTP hydrolysis.     

Domain IV of elongation factor G from Thermus thermophilus is strictly required for translocation.

Two truncated variants of elongation factor G from Thermus thermophilus with deletion of its domain IV have been constructed and the mutated genes were expressed in Escherichia coli. The truncated factors were produced in a soluble form and retained a high thermostability. It was demonstrated that mutated factors possessed (1) a reduced affinity to the ribosomes with an uncleavable GTP analog and (2) a specific ribosome-dependent GTPase activity. At the same time, in contrast to the wild-type elongation factor G, they were incapable to promote translocation. The conclusions are drawn that (1) domain IV is not involved in the GTPase activity of elongation factor G, (2) it contributes to the binding of elongation factor G with the ribosome and (3) is strictly required for translocation. These results suggest that domain IV might be directly involved in translocation and GTPase activity of the factor is not directly coupled with translocation.     

The N terminus of ClpB from Thermus thermophilus is not essential for the chaperone activity

ClpB from Thermus thermophilus belongs to the Clp/Hsp100 protein family and reactivates protein aggregates in cooperation with the DnaK chaperone system. The mechanism of protein reactivation and interaction with the DnaK system remains unclear. ClpB possesses two nucleotide binding domains, which are essential for function and show a complex allosteric behavior. The role of the N-terminal domain that precedes the first nucleotide binding domain is largely unknown. We purified and characterized an N-terminal shortened ClpB variant (ClpBDeltaN; amino acids 140-854), which remained active in refolding assays with three different substrate proteins. In addition the N-terminal truncation did not significantly change the nucleotide binding affinities, the nucleotide-dependent oligomerization, and the allosteric behavior of the protein. In contrast casein binding and stimulation of the ATPase activity by kappa-casein were affected. These results suggest that the N-terminal domain is not essential for the chaperone function, does not influence the binding of nucleotides, and is not involved in the formation of intermolecular contacts. It contributes to the casein binding site of ClpB, but other substrate proteins do not necessarily interact with the N terminus. This indicates a substantial difference in the binding mode of kappa-casein that is often used as model substrate for ClpB and other possibly more suitable substrate proteins.     

Identification of acceptor substrate binding subsites +2 and +3 in the amylomaltase from Thermus thermophilus HB8

Glycoside hydrolase family 77 (GH77) belongs to the alpha-amylase superfamily (Clan H) together with GH13 and GH70. GH77 enzymes are amylomaltases or 4-alpha-glucanotransferases, involved in maltose metabolism in microorganisms and in starch biosynthesis in plants. Here we characterized the amylomaltase from the hyperthermophilic bacterium Thermus thermophilus HB8 (Tt AMase). Site-directed mutagenesis of the active site residues (Asp293, nucleophile; Glu340, general acid/base catalyst; Asp395, transition state stabilizer) shows that GH77 Tt AMase and GH13 enzymes share the same catalytic machinery. Quantification of the enzyme's transglycosylation and hydrolytic activities revealed that Tt AMase is among the most efficient 4-alpha-glucanotransferases in the alpha-amylase superfamily. The active site contains at least seven substrate binding sites, subsites -2 and +3 favoring substrate binding and subsites -3 and +2 not, in contrast to several GH13 enzymes in which subsite +2 contributes to oligosaccharide binding. A model of a maltoheptaose (G7) substrate bound to the enzyme was used to probe the details of the interactions of the substrate with the protein at acceptor subsites +2 and +3 by site-directed mutagenesis. Substitution of the fully conserved Asp249 with a Ser in subsite +2 reduced the activity 23-fold (for G7 as a substrate) to 385-fold (for maltotriose). Similar mutations reduced the activity of alpha-amylases only up to 10-fold. Thus, the characteristics of acceptor subsite +2 represent a main difference between GH13 amylases and GH77 amylomaltases.     

A cytochrome c encoded by the nar operon is required for the synthesis of active respiratory nitrate reductase in Thermus thermophilus

The pgr1 mutant of Arabidopsis thaliana carries a single point mutation (P194L) in the Rieske subunit of the cytochrome b6/f (cyt b6/f) complex and is characterised by a reduced electron transport activity at saturating light intensities in vivo. We have investigated the electron transport in this mutant under in vitro conditions. Measurements of P700 reduction kinetics and of photosynthetic electron transport rates indicated that electron transfer from cyt b6/f to photosystem I is not generally reduced in the mutant, but that the pH dependence of this reaction is altered. The data imply that the pH-dependent inactivation of electron transport through cyt b6/f is shifted by about 1 pH unit to more alkaline pH values in pgr1 thylakoids in comparison with wild-type thylakoids. This interpretation was confirmed by determination of the transmembrane deltapH at different stromal pH values showing that the lumen pH in pgr1 mutant plants cannot drop below pH 6 reflecting most likely a shift of the pK and/or the redox potential of the oxidised Rieske protein.     

Store-operated Ca2+ entry is not required for fertilization-induced Ca2+ signaling in mouse eggs

Repetitive oscillations in cytoplasmic Ca²⁺ due to periodic Ca²⁺ release from the endoplasmic reticulum (ER) drive mammalian embryo development following fertilization. Influx of extracellular Ca²⁺ to support the refilling of ER stores is required for sustained Ca²⁺ oscillations, but the mechanisms underlying this Ca²⁺ influx are controversial. Although store-operated Ca²⁺ entry (SOCE) is an appealing candidate mechanism, several groups have arrived at contradictory conclusions regarding the importance of SOCE in oocytes and eggs. To definitively address this question, Ca²⁺ influx was assessed in oocytes and eggs lacking the major components of SOCE, the ER Ca²⁺ sensor STIM proteins, and the plasma membrane Ca²⁺ channel ORAI1. We generated oocyte-specific conditional knockout (cKO) mice for Stim1 and Stim2, and also generated Stim1/2 double cKO mice. Females lacking one or both STIM proteins were fertile and their ovulated eggs displayed normal patterns of Ca²⁺ oscillations following fertilization. In addition, no impairment was observed in ER Ca²⁺ stores or Ca²⁺ influx following store depletion. Similar studies were performed on eggs from mice globally lacking ORAI1; no abnormalities were observed. Furthermore, spontaneous Ca²⁺ influx was normal in oocytes from Stim1/2 cKO and ORAI1-null mice. Finally, we tested if TRPM7-like channels could support spontaneous Ca²⁺ influx, and found that it was largely prevented by NS8593, a TRPM7-specific inhibitor. Fertilization-induced Ca²⁺ oscillations were also impaired by NS8593. Combined, these data robustly show that SOCE is not required to support appropriate Ca²⁺ signaling in mouse oocytes and eggs, and that TRPM7-like channels may contribute to Ca²⁺ influx that was previously attributed to SOCE.     

Insights into the mode of action of a putative zinc transporter CzrB in Thermus thermophilus

The crystal structures of the cytoplasmic domain of the putative zinc transporter CzrB in the apo and zinc-bound forms reported herein are consistent with the protein functioning in vivo as a homodimer. NMR, X-ray scattering, and size-exclusion chromatography provide support for dimer formation. Full-length variants of CzrB in the apo and zinc-loaded states were generated by homology modeling with the Zn2+/H+ antiporter YiiP. The model suggests a way in which zinc binding to the cytoplasmic fragment creates a docking site to which a metallochaperone can bind for delivery and transport of its zinc cargo. Because the cytoplasmic domain may exist in the cell as an independent, soluble protein, a proposal is advanced that it functions as a metallochaperone and that it regulates the zinc-transporting activity of the full-length protein. The latter requires that zinc binding becomes uncoupled from the creation of a metallochaperone-docking site on CzrB.     

The CorA Mg2+ channel is required for the virulence of Salmonella enterica serovar typhimurium

CorA is the primary Mg²⁺ channel in Salmonella enterica serovar Typhimurium. A corA mutant is attenuated in mice and defective for invasion of and replication within epithelial cells. Microarray studies show that several virulence effectors are repressed in a corA mutant strain, which ultimately manifests itself as a decrease in virulence.     

Nuclease activity of the MutS homologue MutS2 from Thermus thermophilus is confined to the Smr domain

MutS homologues are highly conserved enzymes engaged in DNA mismatch repair (MMR), meiotic recombination and other DNA modifications. Genome sequencing projects have revealed that bacteria and plants possess a MutS homologue, MutS2. MutS2 lacks the mismatch-recognition domain of MutS, but contains an extra C-terminal region called the small MutS-related (Smr) domain. Sequences homologous to the Smr domain are annotated as ‘proteins of unknown function’ in various organisms ranging from bacteria to human. Although recent in vivo studies indicate that MutS2 plays an important role in recombinational events, there had been only limited characterization of the biochemical function of MutS2 and the Smr domain. We previously established that Thermus thermophilus MutS2 (ttMutS2) possesses endonuclease activity. In this study, we report that a Smr-deleted ttMutS2 mutant retains the dimerization, ATPase and DNA-binding activities, but has no endonuclease activity. Furthermore, the Smr domain alone was stable and functional in binding and incising DNA. It is noteworthy that an endonuclease activity is associated with a MutS homologue, which is generally thought to recognize specific DNA structures.     

The Ras related GTPase Miro is not required for mitochondrial transport in Dictyostelium discoideum

Ras-related GTPases of the Miro family have been implicated in mitochondrial homeostasis and microtubule-dependent transport. They consist of two GTP-binding domains separated by calcium-binding motifs and of a C-terminal transmembrane domain that targets the protein to the outer mitochondrial membrane. We disrupted the single Miro-encoding gene in Dictyostelium discoideum and observed a substantial growth defect that we attribute to a decreased mitochondrial mass and cellular ATP content. However, mutant cells even showed an increased rate of oxygen consumption, while glucose consumption, mitochondrial transmembrane potential and production of reactive oxygen species were unaltered. Processes characteristic of the multicellular stage of the D. discoideum life cycle were also unaltered. Although mitochondria occasionally use microtubules for transport in D. discoideum, their size and distribution were not visibly affected. We found Miro in all branches of the eukaryotic tree with the exception of a few protist lineages (mainly those lacking typical mitochondria). Trypanosomatids and ciliates possess structurally unique homologs lacking the N-terminal or the C-terminal GTPase domain, respectively. We propose that in D. discoideum, as in yeasts and plants, Miro plays roles in mitochondrial homeostasis, but the ability to build a complex that regulates its association to kinesin for microtubule-dependent transport probably arose in metazoans.     

External Mg2+ is required for hyperpolarization to occur in ovulated oocytes of the prawn Palaemon serratus

Immediately after dissection, the ovulated oocyte of the prawn Palaemon serratus had a resting potential E_m of −42 ± 2 mV and a membrane resistance R_m of 15 ± 5 MΩ; the membrane was more permeable to Cl⁻ than to K⁺. The oocyte spontaneously hyperpolarized and E_m gradually reached −70 mV 20–30 min after removal of the oocyte from the female, due to increased membrane permeability to K⁺. However, the hyperpolarization occured only if Mg²⁺ was present in the seawater; external Ca²⁺ was not required. Long-term incubation without external Mg²⁺ depolarized the membrane and increased membrane resistance. After preincubation in Mg²⁺-free ASW, oocytes transferred to standard artificial seawater (ASW) transiently hyperpolarized and then repolarized, before gradually hyperpolarizing to a sustained value of −62 ± mV. The respective roles of external Mg²⁺ and fertilization in eliciting the electrical response of the prawn egg at natural spawning are discussed.     

The F subunit of Thermus thermophilus V1-ATPase promotes ATPase activity but is not necessary for rotation

V(1)-ATPase from the thermophilic bacterium Thermus thermophilus is a molecular rotary motor with a subunit composition of A(3)B(3)DF, and its central rotor is composed of the D and F subunits. To determine the role of the F subunit, we generated an A(3)B(3)D subcomplex and compared it with A(3)B(3)DF. The ATP hydrolyzing activity of A(3)B(3)D (V(max) = 20 s(-1)) was lower than that of A(3)B(3)DF (V(max) = 31 s(-1)) and was more susceptible to MgADP inhibition during ATP hydrolysis. A(3)B(3)D was able to bind the F subunit to form A(3)B(3)DF. The C-terminally truncated F((Delta85-106)) subunit was also bound to A(3)B(3)D, but the F((Delta69-106)) subunit was not, indicating the importance of residues 69-84 of the F subunit for association with A(3)B(3)D. The ATPase activity of A(3)B(3)DF((Delta85-106)) (V(max) = 24 s(-1)) was intermediate between that of A(3)B(3)D and A(3)B(3)DF. A single molecule experiment showed the rotation of the D subunit in A(3)B(3)D, implying that the F subunit is a dispensable component for rotation itself. Thus, the F subunit binds peripherally to the D subunit, but promotes V(1)-ATPase catalysis.     

Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp.

Genetic transformation of auxotrophs of the extreme thermophile Thermus thermophilus HB27 to prototrophy was obtained at high frequencies of 10(-2) to 10(-1) when proliferating cell populations were exposed to chromosomal DNA from a nutritionally independent wild-type strain. The transformation frequency was proportional to the DNA concentration from 10 pg/ml to 100 ng/ml. T. thermophilus HB27 cells did not require chemical treatment to induce competence, although optimal transformation was obtained by the addition of a divalent cation (Ca2+ or Mg2+). Competence was maintained throughout the growth phase, with the highest transformation frequencies at pH 6 to 9 and at 70 degrees C. T. thermophilus HB27 and four other typical Thermus strains, T. thermophilus HB8, T. flavus AT62, T. caldophilus GK24, and T. aquaticus YT1, were also transformed to streptomycin resistance by DNA from their own spontaneous streptomycin-resistant mutants. A cryptic plasmid, pTT8, from T. thermophilus HB8 was introduced into T. thermophilus HB27 Pro- at a frequency of 10(-2).