Michal Zolkiewski

ClpB Cooperates with DnaK, DnaJ, and GrpE in Suppressing Protein Aggregation A NOVEL MULTI-CHAPERONE SYSTEM FROM ESCHERICHIA COLI

ClpB is a heat-shock protein fromEscherichia coli with an unknown function. We studied a possible molecular chaperone activity of ClpB in vitro. Firefly luciferase was denatured in urea and then diluted into the refolding buffer (in the presence of 5 mm ATP and 0.1 mg/ml bovine serum albumin). Spontaneous reactivation of luciferase was very weak (less than 0.02% of the native activity) because of extensive aggregation. Conventional chaperone systems (GroEL/GroES and DnaK/DnaJ/GrpE) or ClpB alone did not reactivate luciferase under those conditions. However, ClpB together with DnaK/DnaJ/GrpE greatly enhanced the luciferase activity regain (up to 57% of native activity) by suppressing luciferase aggregation. This coordinated function of ClpB and DnaK/DnaJ/GrpE required ATP hydrolysis, although the ClpB ATPase was not activated by native or denatured luciferase. When the chaperones were added to the luciferase refolding solutions after 5–25 min of refolding, ClpB and DnaK/DnaJ/GrpE recovered the luciferase activity from preformed aggregates. Thus, we have identified a novel multi-chaperone system from E. coli, which is analogous to the Hsp104/Ssa1/Ydj1 system from yeast. ClpB is the only known bacterial Hsp100 protein capable of cooperating with other heat-shock proteins in suppressing and reversing protein aggregation.

A camel passes through the eye of a needle: protein unfolding activity of Clp ATPases.

Clp ATPases are protein machines involved in protein degradation and disaggregation. The common structural feature of Clp ATPases is the formation of ring‐shaped oligomers. Recent work has shown that the function of all Clp ATPases is based on an energy‐dependent threading of substrates through the narrow pore at the centre of the ring. This review gives an outline of known mechanistic principles of threading machines that unfold protein substrates either before their degradation (ClpA, ClpX, HslU) or during their reactivation from aggregates (ClpB). The place of Clp ATPases within a broad AAA+ superfamily of ATPases associated with various cellular activities suggests that similar mechanisms can be used by other protein machines to induce conformational rearrangements in a wide variety of substrates.

Characterization of human torsinA and its dystonia-associated mutant form

Deletion of a single glutamate in torsinA correlates with early-onset dystonia, the most severe form of a neurological disorder characterized by uncontrollable muscle contractions. TorsinA is targeted to the ER (endoplasmic reticulum) in eukaryotic cells. We investigated the processing and membrane association of torsinA and the dystonia-associated Glu-deletion mutant (torsinAdeltaE). We found that the signal sequence of torsinA (residues 1-20 from the 40 amino-acid long N-terminal hydrophobic region) is cleaved in Drosophila S2 cells, as shown by the N-terminal sequencing after partial protein purification. TorsinA is not secreted from S2 cells. Consistently, sodium carbonate extraction and Triton X-114 treatment showed that torsinA is associated with the ER membrane in CHO (Chinese-hamster ovary) cells. In contrast, a variant of torsinA that contains the native signal sequence without the hydrophobic region Ile24-Pro40 does not associate with the membranes in CHO cells, and a truncated torsinA without the 40 N-terminal amino acids is secreted in the S2 culture. Thus the 20-amino-acid-long hydrophobic segment in torsinA, which remains at the N-terminus after signal-peptide cleavage, is responsible for the membrane anchoring of torsinA. TorsinAdeltaE showed similar cleavage of the 20 N-terminal amino acids and membrane association properties similar to wild-type torsinA but, unlike the wild-type, torsinAdeltaE was not secreted in the S2 culture even after deletion of the membrane-anchoring segment. This indicates that the dystonia-associated mutation produces a structurally distinct, possibly misfolded, form of torsinA, which cannot be properly processed in the secretory pathway of eukaryotic cells.

Nucleotide-induced switch in oligomerization of the AAA+ ATPase ClpB

ClpB is a member of the bacterial protein‐disaggregating chaperone machinery and belongs to the AAA+ superfamily of ATPases associated with various cellular activities. The mechanism of ClpB‐assisted reactivation of strongly aggregated proteins is unknown and the oligomeric state of ClpB has been under discussion. Sedimentation equilibrium and sedimentation velocity show that, under physiological ionic strength in the absence of nucleotides, ClpB from Escherichia coli undergoes reversible self‐association that involves protein concentration‐dependent populations of monomers, heptamers, and intermediate‐size oligomers. Under low ionic strength conditions, a heptamer becomes the predominant form of ClpB. In contrast, ATPγS, a nonhydrolyzable ATP analog, as well as ADP stabilize hexameric ClpB. Consistently, electron microscopy reveals that ring‐type oligomers of ClpB in the absence of nucleotides are larger than those in the presence of ATPγS. Thus, the binding of nucleotides without hydrolysis of ATP produces a significant change in the self‐association equilibria of ClpB: from reactions supporting formation of a heptamer to those supporting a hexamer. Our results show how ClpB and possibly other related AAA+ proteins can translate nucleotide binding into a major structural transformation and help explain why previously published electron micrographs of some AAA+ ATPases detected both six‐ and sevenfold particle symmetry.

Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V.

Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross‐links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross‐links, protein flexibility in the vicinity of a cross‐link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor‐V (CMTI‐V; 7 kD) by differential scanning calorimetry. CMTI‐V offers the advantage of a large, flexible, and solvent‐exposed loop not involved in extensive intra‐molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (Tm), an indicator of native state stabilization, for CMTI‐V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide‐removed C3S/C48S (ΔΔGd50°C = −4 kcal/mole; ΔTm = −22°C), the native state is destabilized more than the denatured state; this also applies to the less‐stable CMTI‐V* (ΔΔGd50°C = −3 kcal/mole; ΔTm = −11°C), in which the disulfide‐containing loop is opened by specific hydrolysis of the Lys44‐Asp45 peptide bond; (2) In the less stable, disulfide‐inserted E38C/W54C (ΔΔGd50°C = −1 kcal/mole; ΔTm = +2°C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide‐engineered V42C/R52C (ΔΔGd50°C = +1 kcal/mole; ΔTm = +17°C), the native state is more stabilized than the denatured state. These results show that a cross‐link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen bonds in the same flexible region of CMTI‐V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross‐link on the denatured state of CMTI‐V was estimated directly by means of a four‐state thermodynamic cycle consisting of native and denatured states of CMTI‐V and CMTI‐V*. Overall, the results show that an enthalpy‐entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross‐link, and residual structure in the denatured state.

Reconfiguration of the proteasome during chaperone-mediated assembly

The proteasomal ATPase ring, comprising Rpt1–Rpt6, associates with the heptameric α-ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt carboxy-terminal tails inserting into pockets of the α-ring. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit. Here we report that the base subassembly of the Saccharomyces cerevisiae proteasome, which includes the Rpt ring, forms a high-affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6 and Rpn14. Chaperone-mediated dissociation was abrogated by a non-hydrolysable ATP analogue, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α-pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3-pocket. Although the Rpt6 tail is not visualized within an α-pocket in mature proteasomes, it inserts into the α2/α3-pocket in the base–CP complex and is important for complex formation. Thus, the Rpt–CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme.

The N-terminal domain of Escherichia coli ClpB enhances chaperone function

ClpB/Hsp104 collaborates with the Hsp70 system to promote the solubilization and reactivation of proteins that misfold and aggregate following heat shock. In Escherichia coli and other eubacteria, two ClpB isoforms (ClpB95 and ClpB80) that differ by the presence or absence of a highly mobile 149‐residues long N‐terminus domain are synthesized from the same transcript. Whether and how the N‐domain contributes to ClpB chaperone activity remains controversial. Here, we show that, whereas fusion of a 20‐residues long hexahistidine extension to the N‐terminus of ClpB95 interferes with its in vivo and in vitro activity, the same tag has no detectable effect on ClpB80 function. In addition, ClpB95 is more effective than ClpB80 at restoring the folding of the model protein preS2‐β‐galactosidase as stress severity increases, and is superior to ClpB80 in improving the high temperature growth and low temperature recovery of dnaK756 ΔclpB cells. Our results are consistent with a model in which the N‐domain of ClpB95 maximizes substrate processing under conditions where the cellular supply of free DnaK–DnaJ is limiting.

Conserved amino acid residues within the amino-terminal domain of ClpB are essential for the chaperone activity.

ClpB from Escherichia coli is a member of a protein-disaggregating multi-chaperone system that also includes DnaK, DnaJ, and GrpE. The sequence of ClpB contains two ATP-binding domains that are enclosed between the amino-terminal and carboxyl-terminal regions. The N-terminal sequence region does not contain known functional sequence motifs. Here, we performed site-directed mutagenesis of four polar residues within the N-terminal domain of ClpB (Thr7, Ser84, Asp103 and Glu109). These residues are conserved in several ClpB homologs. We found that the mutations, T7A, S84A, D103A, and E109A did not significantly affect the secondary structure and thermal stability of ClpB, nor did they inhibit the self-association of ClpB, its basal ATPase activity, or the enhanced rate of the ATP hydrolysis by ClpB in the presence of poly-L-lysine. We observed, however, that three mutations, T7A, D103A, and E109A, reduced the casein-induced activation of the ClpB ATPase. The same three mutant ClpB variants also showed low chaperone activity in the luciferase reactivation assay. We found, however, that the four ClpB mutants, as well as the wild-type, bound similar amounts of inactivated luciferase. In summary, we have identified three essential amino acid residues within the N-terminal region of ClpB that participate in the coupling between a protein-binding signal and the ATP hydrolysis, and also support the chaperone activity of ClpB.

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Hexameric AAA+ ATPases induce conformational changes in a variety of macromolecules. AAA+ structures contain the nucleotide‐binding P‐loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA+ sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA+ ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker‐A Thr with Asn in ClpB, a bacterial AAA+ chaperone which reactivates aggregated proteins. We found that the T‐to‐N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP‐bound conformation and the high‐affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker‐A Thr in sensing the nucleotides γ‐phosphate and in maintaining an allosteric linkage between the P‐loop and the aggregate binding site of ClpB. We postulate that AAA+ ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate‐remodeling activity.

Synergistic Cooperation between Two ClpB Isoforms in Aggregate Reactivation

Bacterial AAA+ ATPase ClpB cooperates with DnaK during reactivation of aggregated proteins. The ClpB-mediated disaggregation is linked to translocation of polypeptides through the channel in the oligomeric ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and ClpB80, which does not contain the substrate-interacting N-terminal domain. The biological role of the truncated isoform ClpB80 is unknown. We found that resolubilization of aggregated proteins in Escherichia coli after heat shock and reactivation of aggregated proteins in vitro and in vivo occurred at higher rates in the presence of ClpB95 with ClpB80 than with ClpB95 or ClpB80 alone. Combined amounts of ClpB95 and ClpB80 bound to aggregated substrates were similar to the amounts of either ClpB95 or ClpB80 bound to the substrates in the absence of another isoform. The ATP hydrolysis rate of ClpB95 with ClpB80, which is linked to the rate of substrate translocation, was not higher than the rates measured for the isolated ClpB95 or ClpB80. We postulate that a reaction step that takes place after substrate binding to ClpB and precedes substrate translocation is rate-limiting during aggregate reactivation, and its efficiency is enhanced in the presence of both ClpB isoforms. Moreover, we found that ClpB95 and ClpB80 form hetero-oligomers, which are similar in size to the homo-oligomers of ClpB95 or ClpB80. Thus, the mechanism of functional cooperation of the two isoforms of ClpB may be linked to their heteroassociation. Our results suggest that the functionality of other AAA+ ATPases may be also optimized by interaction and synergistic cooperation of their isoforms.