[Structure, function and mechanisms of action of ATPases from the AAA superfamily of proteins]. / Struktura funkcja oraz mechanizmy dzialania ATPaz z rodziny AAA.

AAA ATPases are found in all living organisms. Their common feature is the presence of a highly conserved the AAA domain referred to as AAA module that is responsible for ATP binding and hydrolysis. The AAA domain is required for proper function of AAA proteins. It contains 200-250 residues, among them there are two classical motifs, Walker A (GX4GKT) and Walker B (HyDE). AAA proteins participate in variety of cellular processes, including cell-cycle regulation, protein proteolysis and disaggregation, organelle biogenesis and intracellular transport. Some of them function as molecular chaperones, subunits of proteolytic complexes or independent proteases (FtsH, Lon). They also act as DNA helicases and transcription factors. This review describes the structure, function and mechanisms of action of the most known AAA proteins.

Interactions within the ClpB/DnaK bi-chaperone system from Escherichia coli.

ClpB and DnaK form a bi-chaperone system that reactivates strongly aggregated proteins in vivo and in vitro. Previously observed interaction between purified ClpB and DnaK suggested that one of the chaperones might recruit its partner during substrate reactivation. We show that ClpB from Escherichia coli binds at the substrate binding site of DnaK and the interaction is supported by the N-terminal domain and the middle domain of ClpB. Moreover, the interaction between ClpB and DnaK depends on the nucleotide-state of DnaK: it is stimulated by ADP and inhibited by ATP. These observations indicate that DnaK recognizes selected structural motifs in ClpB as "pseudo-substrates" and that ClpB may compete with bona fide substrates of DnaK. We conclude that direct interaction between ClpB and DnaK does not mediate a substrate transfer between the chaperones, it may, however, play a role in the recruitment of the bi-chaperone system to specific recognition sites in aggregated particles.

[Role of Escherichia coli molecular chaperones in the protection of bacterial cells against irreversible aggregation induced by heat shock]. / Rola bialek opiekunczych Escherichia coli w ochronie komórki bakteryjnej przed nieodwracalna agregacja bialek indukowana termicznie.

All living organisms respond to environmental stresses, such as heat or ethanol by increasing the synthesis of a specific group of proteins termed heat shock proteins (Hsps) or stress proteins. Major Hsps are molecular chaperones and proteases. Molecular chaperones facilitate the proper folding of polypeptides, protect other proteins from inactivation, and reactivate aggregated proteins. Heat shock proteases eliminate proteins irreversibly damaged by stress. This review describes the role of heat shock proteins of the model bacterial cell, E. coli in the protection of other proteins against aggregation and in the mechanism of removal of protein aggregates from the cell. This mechanism remains unclear and it is believed to involve substrate renaturation and proteolysis by molecular chaperones and heat shock proteases. Recently, many studies have been focused on the disaggregation and reactivation of proteins by a bi-chaperone system consisting of DnaK/DnaJ/GrpE and ClpB, an ATPase from the AAA superfamily of proteins.

The amino-terminal domain of ClpB supports binding to strongly aggregated proteins.

Bacterial heat-shock proteins, ClpB and DnaK form a bichaperone system that efficiently reactivates aggregated proteins. ClpB undergoes nucleotide-dependent self-association and forms ring-shaped oligomers. The ClpB-assisted dissociation of protein aggregates is linked to translocation of substrates through the central channel in the oligomeric ClpB. Events preceding the translocation step, such as recognition of aggregates by ClpB, have not yet been explored, and the location of the aggregate-binding site in ClpB has been under discussion. We investigated the reactivation of aggregated glucose-6-phosphate dehydrogenase (G6PDH) by ClpB and its N-terminally truncated variant ClpBDeltaN in the presence of DnaK, DnaJ, and GrpE. We found that the chaperone activity of ClpBDeltaN becomes significantly lower than that of the full-length ClpB as the size of G6PDH aggregates increases. Using a "substrate trap" variant of ClpB with mutations of Walker B motifs in both ATP-binding modules (E279Q/E678Q), we demonstrated that ClpBDeltaN binds to G6PDH aggregates with a significantly lower affinity than the full-length ClpB. Moreover, we identified two conserved acidic residues at the surface of the N-terminal domain of ClpB that support binding to G6PDH aggregates. Those N-terminal residues (Asp-103, Glu-109) contribute as much substrate-binding capability to ClpB as the conserved Tyr located at the entrance to the ClpB channel. In summary, we provided evidence for an essential role of the N-terminal domain of ClpB in recognition and binding strongly aggregated proteins.

Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells.

Submission of wild-type Escherichia coli to heat shock causes an aggregation of cellular proteins. The aggregates (S fraction) are separable from membrane fractions by ultracentrifugation in a sucrose density gradient. In contrast, no protein aggregation was detectable in an E. coli grpE280 mutant either by this technique or by electron microscopy. In search of an explanation for this observation at a molecular level, two kinds of marker proteins were used: Fda (fructose-1,6-biphosphate aldolase), the previously identified S fraction component, and IbpA/B, small heat-shock proteins abundantly associated with the S fraction proteins. Both types of marker proteins, normally never found in the outer-membrane (OM) fraction of WT cells, were present in the OM fraction from grpE cells after heat shock. This pointed to the presence of aggregates smaller than those in WT cells that cosedimented with the OM fraction. The OM fraction was enlarged in grpE cells. Although not proven directly, the presence of still smaller aggregates, not exceeding the solubility level and containing inactive Fda, was noted in the soluble CP fraction containing the cytoplasmic and periplasmic proteins. Therefore, aggregation occurred in both strains, but in a different way. The autoregulation of the heat-shock response causes a greater increase of DnaK/DnaJ and IbpAB levels in grpE cells than in WT after temperature elevation. This may explain the prevalence of the small-sized aggregates in the grpE cells. Estimation of total Fda protein before and after heat shock did not show any loss. This indicated that renaturation rather than proteolysis underlies the final disappearance of the aggregates. Though surprising at first, this is not contradictory with the participation of heat-shock proteases in removal of protein components of the S fraction as shown before, since proteins that are irreversibly denatured are probably substrates for the proteases.

Structure and function of the middle domain of ClpB from Escherichia coli.

ClpB belongs to the Hsp100/Clp ATPase family. Whereas a homologue of ClpB, ClpA, interacts with and stimulates the peptidase ClpP, ClpB does not associate with peptidases and instead cooperates with DnaK/DnaJ/GrpE in an efficient reactivation of severely aggregated proteins. The major difference between ClpA and ClpB is located in the middle sequence region (MD) that is much longer in ClpB than in ClpA and contains several segments of coiled-coil-like heptad repeats. The function of MD is unknown. We purified the isolated MD fragment of ClpB from Escherichia coli (residues 410-570). Circular dichroism (CD) detected a high population of alpha-helical structure in MD. Temperature-induced changes in CD showed that MD is a thermodynamically stable folding domain. Sedimentation equilibrium showed that MD is monomeric in solution. We produced four truncated variants of ClpB with deletions of the following heptad-repeat-containing regions in MD: 417-455, 456-498, 496-530, and 531-569. We found that the removal of each heptad-repeat region within MD strongly inhibited the oligomerization of ClpB, which produced low ATPase activity of the truncated ClpB variants as well as their low chaperone activity in vivo. Only one ClpB variant (Delta417-455) could partially complement the growth defect of the clpB-null E. coli strain at 50 degrees C. Our results show that heptad repeats in MD play an important role in stabilization of the active oligomeric form of ClpB. The heptad repeats are likely involved in stabilization of an intra-MD helical bundle rather than an intersubunit coiled coil.