Hans-Joachim Schönfeld

A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor sigma32.

The chaperone system formed by DnaK, DnaJ and GrpE mediates stress‐dependent negative modulation of the Escherichia coli heat shock response, probably through association with the heat shock promoter‐specific sigma32 subunit of RNA polymerase. Interactions of the DnaK system with sigma32 were analysed. DnaJ and DnaK bind free, but not RNA polymerase‐bound, sigma32 with dissociation constants of 20 nM and 5 muM respectively. Association and dissociation rates of DnaJ‐sigma32 complexes are 5900‐ and 20‐fold higher respectively than those of DnaK‐sigma32 complexes in the absence of ATP. ATP destabilizes DnaK‐sigma32 interactions. DnaJ, through rapid association with sigma32 and stimulation of hydrolysis of DnaK‐bound ATP, mediates efficient binding of DnaK to sigma32 in the presence of ATP, resulting in DnaK‐DnaJ‐sigma32 complexes containing ADP. GrpE binding to these complexes stimulates nucleotide release and subsequent complex dissociation by ATP. We propose that the principles of this cycle also operate in other chaperone activities of the DnaK system. DnaK and DnaJ cooperatively inhibit sigma32 activity in heat shock gene transcription and GrpE partially reverses this inhibition. These data indicate that reversible inhibition of sigma32 activity through transient association of DnaK and DnaJ is a central regulatory element of the heat shock response.

SUBSTRATE SPECIFICITY OF THE SECB CHAPERONE

The bacterial chaperone SecB assists translocation of proteins across the inner membrane. The mechanism by which it differentiates between secretory and cytosolic proteins is poorly understood. To identify its binding motif, we screened 2688 peptides covering sequences of 23 proteins for SecB binding. The motif is ∼9 residues long and is enriched in aromatic and basic residues, whereas acidic residues are disfavored. Its identification allows the prediction of binding regions within protein sequences with up to 87% accuracy. SecB-binding regions occur statistically every 20–30 residues. The occurrence and affinity of binding regions are similar in SecB-dependent and -independent secretory proteins and in cytosolic proteins, and SecB lacks specificity toward signal sequences. SecB cannot thus differentiate between secretory and non-secretory proteins via its binding specificity. This conclusion is supported by the finding that SecB binds denatured luciferase, thereby allowing subsequent refolding by the DnaK system. SecB may rather be a general chaperone whose involvement in translocation is mediated by interactions of SecB and signal sequences of SecB-bound preproteins with the translocation apparatus.

Sequential action of two hsp70 complexes during protein import into mitochondria

The mitochondrial chaperone mhsp70 mediates protein transport across the inner membrane and protein folding in the matrix. These two reactions are effected by two different mhsp70 complexes. The ADP conformation of mhsp70 favors formation of a complex on the inner membrane; this ‘import complex’ contains mhsp70, its membrane anchor Tim44 and the nucleotide exchange factor mGrpE. The ATP conformation of mhsp70 favors formation of a complex in the matrix; this ‘folding complex’ contains mhsp70, the mitochondrial DnaJ homolog Mdj1 and mGrpE. A precursor protein entering the matrix interacts first with the import complex and then with the folding complex. A chaperone can thus function as part of two different complexes within the same organelle.

Genetic Analysis and Functional Characterization of the Streptococcus pneumoniae vic Operon

ABSTRACT The vic two-component signal transduction system of Streptococcus pneumoniae is essential for growth. The vic operon comprises three genes encoding the following: VicR, a response regulator of the OmpR family; VicK, its cognate histidine kinase; and VicX, a putative protein sharing 55% identity to the predicted product (YycJ) of an open reading frame in the Bacillus subtilis genome. We show that not only is vic essential for viability but it also influences virulence and competence. A putative transcriptional start site for the vic operon was mapped 16 bp upstream of the ATG codon of vicR. Only one transcript of 2.9 kb, encoding all three genes, was detected by Northern blot analysis. VicK, an atypical PAS domain-containing histidine kinase, can be autophosphorylated in vitro, and VicR functions in vitro as a phospho-acceptor protein. (PAS is an acronym formed from the names of the proteins in which the domains were first recognized: the Drosophila period clock protein [PER], vertebrate aryl hydrocarbon receptor nuclear translocator [ARNT], and Drosophila single-minded protein [SIM].) PAS domains are commonly involved in sensing intracellular signals such as redox potential, which suggests that the signal for vic might also originate in the cytoplasm. Growth rate, competence, and virulence were monitored in strains with mutations in the vic operon. Overexpression of the histidine kinase, VicK, resulted in decreased virulence, whereas the transformability of a null mutant decreased by 3 orders of magnitude.

Three-state equilibrium of Escherichia coli trigger factor

Abstract Trigger Factor (TF) is the first chaperone that interacts with nascent chains of cytosolic proteins in Escherichia coli. Although its chaperone activity requires association with ribosomes, TF is present in vivo in a 2 3 fold molar excess over ribosomes and a fraction of it is not ribosomeassociated after cell lysis. Here we show that TF follows a threestate equilibrium. Size exclusion chromatography, crosslinking and analytical ultracentrifugation revealed that uncomplexed TF dimerizes with an apparent Kd of 18 M. Dimerization is mediated by the Nterminal ribosome binding domain and the Cterminal domain of TF, whereas the central peptidyl prolyl isomerase (PPIase) and substrate binding domain does not contribute to dimerization. Crosslinking experiments showed that TF is monomeric in its ribosomeassociated state. Quantitative analysis of TF binding to ribosomes revealed a dissociation constant for the TFribosome complex of approximately 1.2 M. From these data we estimate that in vivo most of the ribosomes are in complex with monomeric TF. Uncomplexed TF, however, is in a monomerdimer equilibrium with approximately two thirds of TF existing in a dimeric state.

The HspR regulon of Streptomyces coelicolor: a role for the DnaK chaperone as a transcriptional co‐repressor†

The dnaK operon of Streptomyces coelicolor encodes the DnaK chaperone machine and HspR, the transcriptional repressor of the operon; HspR confers repression by binding to several inverted repeat sequences in the promoter region, dnaKp. Here, we demonstrate that HspR specifically requires the presence of DnaK protein to retard a dnaKp fragment in gel‐shift assays. This requirement is independent of the co‐chaperones, DnaJ and GrpE, and it is ATP independent. Furthermore the retarded protein–DNA complex can be ‘supershifted’ by anti‐DnaK monoclonal antibody, demonstrating that DnaK forms an integral component of the complex. It was shown in DNase I footprinting experiments that refolding and specific binding of HspR to its DNA target does not require DnaK. We conclude that the formation of the stable DnaK–HspR–DNA ternary complex does not depend on the chaperoning activity of DnaK. In affinity chromatography experiments using whole‐cell extracts, DnaK was shown to co‐purify with HspR, providing additional evidence that the two proteins interact in vivo; it was not possible to purify HspR away from DnaK in any experiments unless a powerful denaturant was used. The level of heat shock induction of chromosomal DnaK could be partially suppressed by expressing dnaK extrachromosomally from a heterologous promoter. In addition, it is shown that DnaK confers enhanced HspR‐mediated repression of transcription in vitro. Taken together, these results suggest that DnaK functions as a transcriptional co‐repressor by binding to HspR at its operator sites. In this model, the DnaK–HspR system would represent a novel example of feedback regulation of gene expression by a molecular chaperone, in which DnaK directly activates a repressor, rather than inactivates an activator (as is the case in the DnaK–σ32 and Hsp70–HSF systems of other organisms).

d-Peptide Ligands for the Co-chaperone DnaJ

The molecular chaperone DnaK, the Hsp70 homolog of Escherichia coli, binds hydrophobic polypeptide segments in extended conformation. The co-chaperone DnaJ (Hsp40) has been reported to bind native and denatured proteins as well as peptides. We tested pseudo-peptides of d-amino acids as ligands for both chaperones. In comparison to the parent all- lpeptide, these mimetics had either enantiomorphic side chain positions combined with retained main chain direction (normal all- d peptide) or unchanged side chain topology together with reverse direction of the peptide backbone (retro all- d peptide). The peptides were labeled with acrylodan (a), and their binding to DnaK and DnaJ was monitored by the accompanying increase in fluorescence intensity. The parent all- l peptide a-CALLLSAARR bound to both DnaK (Kd = 0.1 μm) and DnaJ (Kd = 9.2 μm). In contrast, the normal all- dand retro all-d peptides did not bind to DnaK; they bound, however, to DnaJ withKd values of 6.8 μm and 0.9 μm, respectively. The emission spectra of the DnaJ-bound peptides suggests that DnaJ bound both d-peptides with the same main chain direction as l-peptides. Binding of thenormal all- d and all- lpeptides inhibited the DnaJ-induced stimulation of DnaK ATPase. However, binding of the retro all-d analog to DnaJ did not impair the stimulation, indicating the existence of separate binding sites for peptides and DnaK.

Altered differentiation of limb bud cells by transforming growth factors-β isolated from bone matrix and from platelets

A crude extract of demineralized bone matrix caused an altered differentiation of limb bud cells which was seen within 5 days in culture. Using this bioassay system we purified two factors to homogeneity and found that according to their N-terminal sequences they corresponded to TGF-beta 1 and TGF-beta 2 isolated from platelets. Biochemical analyses and biological studies (molecular mass determination, inactivation by reducing agents and proteases, antibody neutralization, competitive binding to TGF-beta receptors and influence on protein expression) provided additional evidence that the two proteins isolated from demineralized bone matrix were apparently identical to TGF-beta 1 and TGF-beta 2. Proteoglycan content, alkaline phosphatase activity and response of the cells to PTH stimulated adenylate cyclase were quantitatively changed by the factors. Culturing limb bud cells on polycarbonate membranes resulted in a rapid and extensive growth and differentiation of the cells to palpable tissue pieces. Relative to controls distinct cell and tissue morphology was observed macroscopically and in histological sections of these tissue pieces.