Structure-function analysis of the zinc-binding region of the Clpx molecular chaperone.

The ClpX heat shock protein of Escherichia coli is a member of the universally conserved Hsp100 family of proteins, and possesses a putative zinc finger motif of the C(4) type. The ClpX is an ATPase which functions both as a substrate specificity component of the ClpXP protease and as a molecular chaperone. Using an improved purification procedure we show that the ClpX protein is a metalloprotein complexed with Zn(II) cations. Contrary to other Hsp100 family members, ClpXZn(II) exists in an oligomeric form even in the absence of ATP. We show that the single ATP-binding site of ClpX is required for a variety of tasks, namely, the stabilization of the ClpXZn(II) oligomeric structure, binding to ClpP, and the ClpXP-dependent proteolysis of the lambdaO replication protein. Release of Zn(II) from ClpX protein affects the ability of ClpX to bind ATP. ClpX, free of Zn(II), cannot oligomerize, bind to ClpP, or participate in ClpXP-dependent proteolysis. We also show that ClpXDeltaCys, a mutant protein whose four cysteine residues at the putative zinc finger motif have been replaced by serine, behaves in similar fashion as wild type ClpX protein whose Zn(II) has been released either by denaturation and renaturation, or chemically by p-hydroxymercuriphenylsulfonic acid.

Role of adenine nucleotides, molecular chaperones and chaperonins in stabilization of DnaA initiator protein of Escherichia coli.

DnaA protein of Escherichia coli is a sequence-specific DNA binding protein required for the initiation of DNA replication from the chromosomal origin, oriC, and of several E. coli plasmids. At a moderate ionic strength, purified DnaA protein has a strong tendency to aggregate; the self-aggregate form is inactive in DNA replication. Binding of ATP or ADP to DnaA protein protected it from aggregation to maintain its replication activity. AMP or cyclic AMP had no protective effect. The molecular chaperone DnaK protected DnaA protein from aggregation with or without ATP. DnaJ and GrpE were not stimulatory. Chaperonins GroEL and GroES were also able to prevent aggregation but only in the presence of ATP. The studies presented here show that for DnaA protein to be active in the initiation of DNA replication, it must be prevented from forming a self-aggregate by the binding of adenine nucleotides, and/or by the action of molecular chaperones.

The Clp ATPases define a novel class of molecular chaperones.

The Clp ATPases were originally identified as a regulatory component of the bacterial ATP-dependent Clp serine proteases. Proteins homologous to the Escherichia coli Clp ATPases (ClpA, B, X or Y) have been identified in every organism examined so far. Recent data suggest that the Clp ATPases are not only specificity factors which help to 'present' various protein substrates to the ClpP or other catalytic proteases, but are also molecular chaperones which can function independently of ClpP. This review discusses the recent evidence that the Clp ATPases are indeed molecular chaperones capable of either repairing proteins damaged during stress conditions or activating the initiation proteins for Mu, lambda or P1 DNA replication. A mechanism is suggested to explain how the Clp ATPases 'decide' whether to repair or destroy their protein substrates.

Structure-function analysis of the zinc finger region of the DnaJ molecular chaperone.

DnaJ is a molecular chaperone, which not only binds to its various protein substrates, but can also activate the DnaK cochaperone to bind to its various protein substrates as well. DnaJ is a modular protein, which contains a putative zinc finger motif of unknown function. Quantitation of the released Zn(II) ions, upon challenge with p-hydroxymercuriphenylsulfonic acid, and by atomic absorption showed that two Zn(II) ions interact with each monomer of DnaJ. Following the release of Zn(II) ions, the free cysteine residues probably form disulfide bridge(s), which contribute to overcoming the destabilizing effect of losing Zn(II). Supporting this view, infrared and circular dichroism studies show that the DnaJ secondary structure is largely unaffected by the release of Zn(II). Moreover, infrared spectra recorded at different temperatures, as well as scanning calorimetry, show that the Zn(II) ions help to stabilize DnaJ's tertiary structure. An internal 57-amino acid deletion of the cysteine-reach region did not noticeably affect the affinity of this mutant protein, DnaJDelta144-200, to bind DnaK nor its ability to stimulate DnaK's ATPase activity. However, the DnaJDelta144-200 was unable to induce DnaK to a conformation required for the stabilization of the DnaK-substrate complex. Additionally, the DnaJDelta144-200 mutant protein alone was unimpaired in its ability to interact with its final sigma32 transcription factor substrate, but exhibited reduced affinity toward its P1 RepA and lambdaP substrates. Finally, these in vitro results correlate well with the in vivo observed partial inhibition of bacteriophage lambda growth in a DnaJDelta144-200 mutant background.

Real time kinetics of the DnaK/DnaJ/GrpE molecular chaperone machine action.

Applying stopped-flow fluorescence spectroscopy for measuring conformational changes of the DnaK molecular chaperone (bacterial Hsp70 homologue) and its binding to target peptide, we found that after ATP hydrolysis, DnaK is converted to the DnaK*(ADP) conformation, which possesses limited affinity for peptide substrates and the GrpE cochaperone but efficiently binds the DnaJ chaperone. In the presence of DnaJ (bacterial Hsp40 homologue), the DnaK*(ADP) form is converted back to the DnaK conformation, and the resulting DnaJ-DnaK(ADP) complex binds to peptide substrates more tightly. Formation of the DnaJ(substrate-DnaK(ADP)) complex is a rate-limiting reaction. The presence of GrpE and ATP hydrolysis promotes the fast release of the peptide substrate from the chaperone complex and converts DnaK to the DnaK*(ADP) conformation. We conclude that in the presence of DnaJ and GrpE, the binding-release cycle of DnaK is stoichiometrically coupled to the adenosine triphosphatase activity of DnaK.

ATP hydrolysis is required for the DnaJ-dependent activation of DnaK chaperone for binding to both native and denatured protein substrates.

Using two independent experimental approaches to monitor protein-protein interactions (enzyme-linked immunosorbent assay and size exclusion high performance liquid chromatography) we describe a general mechanism by which DnaJ modulates the binding of the DnaK chaperone to various native protein substrates, e.g. lambda P, lambda O, delta 32, P1, RepA, as well as permanently denatured alpha-carboxymethylated lactalbumin. The presence of DnaJ promotes the DnaK for efficient DnaK-substrate complex formation. ATP hydrolysis is absolutely required for such DnaJ-dependent activation of DnaK for binding to both native and denatured protein substrates. Although ADP can stabilize such as an activated DnaK-protein complex, it cannot substitute for ATP in the activation reaction. In the presence of DnaJ and ATP, DnaK possesses the affinity to different substrates which correlates well with the affinity of DnaJ alone for these protein substrates. Only when the affinity of the DnaJ chaperone for its protein substrate is relatively high (e.g. delta 32, RepA) can a tertiary complex DnaK-substrate-DnaJ be detected. In the case that DnaJ binds weakly to its substrate (lambda P, alpha-carboxymethylated lactalbumin), DnaJ is only transiently associated with the DnaK-substrate complex, but the DnaK activation reaction still occurs, albeit less efficiently.

The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone.

All major classes of protein chaperones, including DnaK (the Hsp70 eukaryotic equivalent) and GroEL (the Hsp60 eukaryotic equivalent) have been found in Escherichia coli. Molecular chaperones enhance the yields of correctly folded polypeptides by preventing aggregation and even by disaggregating certain protein aggregates. Previously, we identified the ClpX heat-shock protein of E. coli because it enables the ClpP catalytic protease to degrade the bacteriophage lambda O replication protein. Here we report that ClpX alone possesses all the properties expected of a molecular chaperone protein. Specifically, it can protect the lambda O protein from heat-induced aggregation, disaggregate preformed lambda O aggregates, and even promote efficient binding of lambda O to its DNA recognition sequence. A lambda O-ClpX specific protein-protein interaction can be detected either by a modified ELISA assay or through the stimulation of ClpX's weak ATPase activity by lambda O. Unlike the behaviour of the major DnaK and GroEL chaperones, ClpX requires the presence of ATP or its non-hydrolysable analogue ATP-gamma-S for efficient interaction with other proteins including the protection of lambda O from aggregation. However, ClpX's ability to disaggregate lambda O aggregates requires hydrolysable ATP. We propose that the ClpX protein is a bona fide chaperone, whose biological role includes the maintenance of certain polypeptides in a form competent for proteolysis by the ClpP protease. Furthermore, our results suggest that the ClpX protein also performs typical chaperone protein functions independent of ClpP.

Structural and functional relationships in DnaK and DnaK756 heat-shock proteins from Escherichia coli.

The secondary structures of DnaK and the mutant DnaK756 heat-shock proteins from Escherichia coli have been investigated by Fourier transform infrared spectroscopy. The analysis of infrared data showed that DnaK and DnaK756 proteins have different secondary structures that are not affected by the presence of ATP or beta, gamma-methyleneadenosine 5'-triphosphate. The infrared data indicate also that the tertiary structures of DnaK and DnaK756 proteins are different and that DnaK protein undergoes conformational changes in its tertiary structure not only during binding of ATP but also during ATP hydrolysis. Using fluorescence spectroscopy of a single tryptophan located in the N-terminal domain of DnaK protein and fluorescence of 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid, which interacts with hydrophobic domains of DnaK protein, we were able to distinguish between two conformational states of DnaK protein. After binding of triphosphonucleotides, the C-terminal domain of DnaK protein changes in tertiary structure in such a way that fewer hydrophobic segments are exposed on the surface of the protein. After ATP hydrolysis, the number of hydrophobic segments on the surface of the protein is further reduced, and moreover the tertiary structure of the N-terminal domain of the protein changes. These data are discussed in terms of structural and functional relationships of both DnaK and DnaK756 proteins.