The Amino-terminal Domain of ClpB Supports Binding to Strongly Aggregated Proteins

Micheal E Barnett,Maria Nagy,Sabina Kedzierska,Michal Zolkiewski

doi:10.1074/jbc.m505653200

Micheal E Barnett, Maria Nagy + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m505653200

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Abstract

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.

Highlights

It has been shown that the mechanism of protein disaggregation mediated by ClpB involves ATP-dependent threading of substrates through the central channel in the oligomeric ring [11], which implies that ClpB binds exposed termini of aggregated polypeptides
That group of amino acids is located within the AAAϩ module and is found in many AAAϩ ATPases, regardless of their function [13]
We produced aggregates of glucose-6-phosphate dehydrogenase (G6PDH) using a procedure analogous to that developed by Goloubinoff and co-workers [27]

Summary

EXPERIMENTAL PROCEDURES

Proteins—Previously published procedures were used to produce and purify the Escherichia coli chaperones, ClpB and ClpB⌬N [17], DnaK [23], and DnaJ [24]. The mixture was incubated at 47 °C for 5 min, at which time 90 ␮l of the refolding buffer (50 mM triethanolamine/Cl, pH 7.5, 20 mM Mg(OAc) mM KCl, 1 mM ␤-mercaptoethanol, and 1 mM EDTA) without or with 2 mM ATP or ADP was added, and the G6PDH sample was mixed vigorously and incubated at 47 °C for a variable period of time. 20 ␮l of 5ϫ SDS loading buffer was added to the pellet, the sample was boiled for 2 min, and analyzed by SDS-PAGE. G6PDH Reactivation Assay—Aggregates of G6PDH (22 ␮M) were diluted 8-fold into the refolding buffer with 5 mM ATP containing no chaperones, 1 ␮M DnaK with 0.5 ␮M DnaJ and 0.5 ␮M GrpE (KJE), KJE with 1.5 ␮M ClpB, or KJE with 1.5 ␮M ClpB⌬N. G6PDH Activity Assay—G6PDH samples were incubated at 30 °C in 50 mM Tris-HCl, pH 7.8, 5 mM MgCl2 with 2 mM glucose-6-phosphate

The abbreviation used is

RESULTS

DISCUSSION