Trigonal DnaK-DnaJ Complex Versus Free DnaK and DnaJ

100

113

Hsp70 is a central molecular chaperone that passively prevents protein aggregation and uses the energy of ATP hydrolysis to solubilize, translocate, and mediate the proper refolding of proteins in the cell. Yet, the molecular mechanism by which the active Hsp70 chaperone functions are achieved remains unclear. Here, we show that the bacterial Hsp70 (DnaK) can actively unfold misfolded structures in aggregated polypeptides, leading to gradual disaggregation. We found that the specific unfolding and disaggregation activities of individual DnaK molecules were optimal for large aggregates but dramatically decreased for small aggregates. The active unfolding of the smallest aggregates, leading to proper global refolding, required the cooperative action of several DnaK molecules per misfolded polypeptide. This finding suggests that the unique ATP-fueled locking/unlocking mechanism of the Hsp70 chaperones can recruit random chaperone motions to locally unfold misfolded structures and gradually disentangle stable aggregates into refoldable proteins.

Section: Passive and Active Molecular Mechanisms Of The Hsp70supporting

confidence: 81%

Section: Passive and Active Molecular Mechanisms Of The Hsp70mentioning

confidence: 99%

Active Solubilization and Refolding of Stable Protein Aggregates By Cooperative Unfolding Action of Individual Hsp70 Chaperones

Ben‐Zvi

Rios

Dietler

et al. 2004

100

113

“…CbpM shares some weak homology with DafA of Thermus thermophilus, a protein found to promote the assembly of a ring-like structure containing a trimer each of DnaK Tth , DnaJ Tth , and DafA Tth (16). The role of DafA Tth in protein folding by DnaK Tth has been explored, and the function of DafA Tth may be to hold DnaK Tth and DnaJ Tth in an inactive state during non-stress conditions (17)(18)(19).…”

mentioning

confidence: 99%

Functional Analysis of CbpA, a DnaJ Homolog and Nucleoid-associated DNA-binding Protein

Bird¹,

Sharma²,

Roshwalb

et al. 2006

DnaK/Hsp70 proteins are universally conserved ATP-dependent molecular chaperones that help proteins adopt and maintain their native conformations. DnaJ/Hsp40 and GrpE are co-chaperones that assist DnaK. CbpA is an Escherichia coli DnaJ homolog. It acts as a multicopy suppressor for dnaJ mutations and functions in vitro in combination with DnaK and GrpE in protein remodeling reactions. CbpA binds nonspecifically to DNA with preference for curved DNA and is a nucleoid-associated protein. The DNA binding and co-chaperone activities of CbpA are modulated by CbpM, a small protein that binds specifically to CbpA. To identify the regions of CbpA involved in the interaction of CbpA with CbpM and those involved in DNA binding, we constructed and characterized deletion and substitution mutants of CbpA. We discovered that CbpA interacted with CbpM through its N-terminal J-domain. We found that the region C-terminal to the J-domain was required for DNA binding. Moreover, we found that the CbpM interaction, DNA binding, and co-chaperone activities were separable; some mutants were proficient in some functions and defective in others.

“…To date, such disaggregation activities have been demonstrated for ClpB and its homologues of yeast, Thermus thermophilus, Escherichia coli, and mitochondria (1)(2)(3)(4)(5)(6)(7)(8). ClpB is a member of AAAϩ protein superfamily, and, similar to other members, it assembles into a hexamer in an ATP-dependent manner (9 -15).…”

mentioning

confidence: 99%

ATP Binding to Nucleotide Binding Domain (NBD)1 of the ClpB Chaperone Induces Motion of the Long Coiled-coil, Stabilizes the Hexamer, and Activates NBD2

Watanabe¹,

Takano

Yoshida

2005

Self Cite

The molecular chaperone ClpB can rescue the heatdamaged proteins from an aggregated state in cooperation with other chaperones. It has two nucleotide binding domains (NBD1 and NBD2) and forms a hexamer ring in a manner dependent on ATP binding to NBD1. In the crystal structure of ClpB with both NBDs filled by nucleotides, the linker between two NBDs forms an 85-Å-long coiled-coil that extends on the outside of the hexamer and leans to NBD1. To probe the possible motion of the coiled-coil, we tested the accessibility of a labeling reagent, fluorescence change of a labeled dye, and crosslinking between the coiled-coil and NBD1 by using the mutants with defective NBD1 or NBD2. The results suggest that the coiled-coil is more or less parallel to the main body of ClpB in the absence of nucleotide and that ATP binding to NBD1 brings it to the leaning position as seen in the crystal structure. This motion results in stabilization of the hexamer form of ClpB and promotion of ATP hydrolysis at NBD2.A molecular chaperone ClpB is unique in its activity to rescue heat-damaged proteins from an aggregated state in cooperation with a trio DnaK chaperone set, DnaK/DnaJ/GrpE. To date, such disaggregation activities have been demonstrated for ClpB and its homologues of yeast, Thermus thermophilus, Escherichia coli,. ClpB is a member of AAAϩ protein superfamily, and, similar to other members, it assembles into a hexamer in an ATP-dependent manner (9 -15). It has two nucleotide binding domains (NBD(s)) 1 in a single polypeptide, NBD1 and NBD2, each containing a set of Walker A and B motifs and having a RecA-like fold in which the nucleotide binding cleft exists between the P-loop subdomain and helical subdomain. In addition, ClpB has a unique inserted 120-amino-acid sequence between two NBDs, and this insertion has been inferred to contribute to the disaggregation activity of ClpB (15, 16). The crystal structure of ClpB from T. thermophilus revealed an unusual structure of the inserted region, an 85-Å-long, straight coiled-coil extending on the outside of the hexamer (17). The coiled-coil is tethered to the main body of ClpB at its middle point, resembling in structure the shape of a two-wing propeller, one wing (wing-1) leaning to NBD1 and another wing (wing-2) away from any other domains (see Fig. 1). This "leaning position" of the coiled-coil seems to be stabilized by the hydrophobic interactions between the wing-1 and the ␣-helical subdomain of NBD1. The coiled-coil structure by itself is stabilized by the leucine zipper-like interaction between two helices. The importance of the position and movement of the coiled-coil on the chaperone activity was demonstrated previously (17). To know further the movement of the coiled-coil in the chaperone function, we examined reactivity and fluorescence change of a fluorescent label and cross-linking of NBD1 and the wing-1 of T. thermophilus ClpB. The contribution of nucleotide occupancy and hydrolysis of the two NBDs to the movement of the coiled-coil was assessed from the results of defe...