The Activation Mechanism of Hsp26 does not Require Dissociation of the Oligomer

doi:10.1016/j.jmb.2005.05.034

Journal of Molecular Biology

Volume 350, Issue 5, 29 July 2005, Pages 1083-1093

https://doi.org/10.1016/j.jmb.2005.05.034 Get rights and content

Small heat shock proteins (sHsps) are molecular chaperones that specifically bind non-native proteins and prevent them from irreversible aggregation. A key trait of sHsps is their existence as dynamic oligomers. Hsp26 from Saccharomyces cerevisiae assembles into a 24mer, which becomes activated under heat shock conditions and forms large, stable substrate complexes. This activation coincides with the destabilization of the oligomer and the appearance of dimers. This and results from other groups led to the generally accepted notion that dissociation might be a requirement for the chaperone mechanism of sHsps.

To understand the chaperone mechanism of sHsps it is crucial to analyze the relationship between chaperone activity and stability of the oligomer. We generated an Hsp26 variant, in which a serine residue of the N-terminal domain was replaced by cysteine. This allowed us to covalently crosslink neighboring subunits by disulfide bonds. We show that under reducing conditions the structure and function of this variant are indistinguishable from that of the wild-type protein. However, when the cysteine residues are oxidized, the dissociation into dimers at higher temperatures is no longer observed, yet the chaperone activity remains unaffected. Furthermore, we show that the exchange of subunits between Hsp26 oligomers is significantly slower than substrate aggregation and even inhibited in the presence of disulfide bonds. This demonstrates that the rearrangements necessary for shifting Hsp26 from a low to a high affinity state for binding non-native proteins occur without dissolving the oligomer.

Introduction

Small heat shock proteins (sHsps) belong to the functionally related class of molecular chaperones that specifically recognize non-native proteins.1, 2, 3 As part of the cellular multi-chaperone network, sHsps possess a substantial binding capacity for protein folding intermediates, which allows them to prevent their irreversible aggregation.4, 5, 6, 7, 8 sHsps are characterized by several structural and functional features. The most significant is the presence of the highly conserved α-crystallin domain in the C-terminal part of the proteins.9, 10 In αA and αB-crystallin, the two major eye lens proteins in vertebrates, these domains are composed of about 80 residues with 60% sequence identity.11, 12 Besides this, sHsps share monomeric masses between 12 kDa and 43 kDa. These monomers associate into oligomeric structures with mostly 12 or 24 subunits. Even larger complexes with up to 50 subunits were observed for α-crystallin.13, 14 The large variability in the length of the N-terminal regions appears to be responsible for the significant variations of the sizes of the oligomers.¹² So far, two X-ray structures of sHsps have been resolved. Hsp16.5 from Methanocaldococcus jannaschii assembles into a 24mer and forms a hollow sphere with octahedral symmetry.¹⁵ Hsp16.9 from wheat forms a 12mer arranged into two hexameric rings.¹⁶ Both structures revealed the α-crystallin domain as the dimeric building block. In addition to the α-crystallin domain, sHsps comprise a flexible, disordered N-terminal region and a C-terminal extension. From structural investigations carried out for various sHsps, including Hsp16.5, α-crystallin and Hsp16.2 from Caenorhabditis elegans, it was proposed that the N-terminal regions are sequestered inside the oligomers.15, 17, 18 Recently it was shown that the N-terminal regions are required for substrate interactions¹⁹ and that they play a role in stabilizing the oligomeric state.20, 21 The C-terminal extensions appear to be moderately conserved throughout the sHsps family. In Hsp16.5, these were found to mediate the self-association into the oligomeric complex via inter-subunit interactions.¹⁵ This was confirmed for various sHsps in which the C-terminal extensions were either removed or mutated.21, 22, 23, 24 Mostly these variants fail to associate into their native quaternary structure. The N-terminal region and the C-terminal extension seem to have partially overlapping functions with respect to the association of the oligomer. Furthermore, both regions are required for efficient chaperone activity20, 24 and recent investigations suggest that oligomerization of sHsps is a prerequisite for their chaperone mechanism.17, 21, 25, 26, 27, 28 sHsps are dynamic structures, which permanently exchange subunits between oligomers.29, 30, 31, 32, 33 Although this dynamic behavior seems to be a common feature, no functional importance could be assigned to it so far and it was speculated that the exchange of subunits is linked to the chaperone mechanism.

Hsp26 is one of the two cytosolic sHsps from Saccharomyces cerevisiae.³⁴ Under physiological conditions, it exists as a hollow sphere of 24 subunits assembled from 12 dimers.35, 36, 37 We have shown previously that Hsp26 efficiently suppresses heat-induced aggregation of substrates in vivo and in vitro and associates into defined, stable complexes with model substrates including citrate synthase (CS) and insulin, indicating promiscuous binding properties.6, 7, 38 The first 30 amino acid residues of the N-terminal domain of Hsp26 are required for substrate interaction and stabilize the oligomeric state.²⁰ Residues 30–95 play a role in the association of the native oligomer.¹⁹ An important functional characteristic is that Hsp26 requires elevated temperatures for activation, as it fails to efficiently suppress the reduction-induced aggregation of insulin at room temperature.³⁸ Coinciding with the temperature-dependent activation, the Hsp26 oligomer was shown to become destabilized and dissociate into dimers.

To gain further insight into the chaperone mechanism of sHsps, we generated a point mutant of Hsp26, in which the residue serine 4 of the N-terminal domain (Hsp26_S4C) was replaced by cysteine. The introduction of this reactive residue allowed covalent cross-linking of neighboring subunits by oxidation. Analysis of the structure and function under reducing conditions showed that Hsp26_S4C behaved indistinguishably from the Hsp26 wild-type protein. Interestingly, when the cysteine residues were oxidized, the Hsp26 oligomer was stabilized and subunit exchange and dissociation into dimers was completely inhibited, yet the chaperone activity remained unaffected. These results show that the rearrangements necessary for activation of Hsp26 can occur without dissolving the oligomer and indicate that the Hsp26 oligomer can interact with substrate proteins.

Section snippets

Structural analysis of reduced and oxidized Hsp26_S4C

sHsps consist of a predominantly β-structured α-crystallin domain,9, 10 an unstructured N-terminal region and a moderately conserved C-terminal extension. Since truncations in the N-terminal regions affect the stability of the oligomeric structure, it was tempting to speculate that the N-terminal regions are involved in inter-subunit interactions. The introduction of artificial disulfide bridges in proteins had been used successfully to analyze the importance of conformational rearrangements in

Discussion

sHsps form oligomeric structures, which bind non-native proteins in a cooperative and efficient manner.³ It appears that this class of molecular chaperones can be divided into constitutively active and temperature-controlled sHsps. Hsp26 is a prototypic temperature-regulated sHsp, as it requires elevated temperatures to display chaperone activity. Previously, we reported that the complex of 24 subunits becomes destabilized upon heat-activation, resulting in the appearance of dimers in

Cloning and purification of Hsp26 and Hsp26_S4C

Structural genes of Hsp26 and the Hsp26 cysteine variants were amplified from genomic yeast DNA using PWO polymerase (Roche, Germany) with the following primers (Forward Hsp26: GAT CCC ATG GGG ATG TCA TTT AAC AGT C, Reverse Hsp26: GAT CGC GGC CGC TTA GTT ACC GTA CGA TTC TTG AGA AG, Forward Hsp26_S4C: GAT CCC ATG GGG TCA TTT AAC TGT CCA TTT TTT GAT TTC). The DNA fragments were ligated into pET28b+ (Novagene) and expressed in Escherichia coli BL21 (DE3) (Stratagene) at 30 °C for four hours.

Acknowledgements

Hsp26ΔN was a kind gift from Thusnelda Stromer. We are indebted to Martin Heβling and Lin Müller for discussions. Bettina Richter is acknowledged for TEM assistance. We thank Helmut Krause and Anja Osterauer for support with mass spectrometry and protein purification. We also thank Birgit Meinlschmidt, Martin Haslbeck and Daniel Weinfurtner for reading the manuscript. This work was funded by the grants of the Deutsche Forschungsgesellschaft to J.B. and S.W.

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Journal of Molecular Biology

The Activation Mechanism of Hsp26 does not Require Dissociation of the Oligomer

Introduction

Section snippets

Structural analysis of reduced and oxidized Hsp26S4C

Discussion

Cloning and purification of Hsp26 and Hsp26S4C

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

Int. J. Biol. Macromol.

Int. J. Biol. Macromol.

J. Biol. Chem.

Expt. Eye Res.

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

FEBS Letters

Comput. Biochem. Physiol. B Biochem. Mol. Biol.

J. Biol. Chem.

FEBS Letters

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Gene

J. Biol. Chem.

J. Biol. Chem.

Expt. Eye Res.

FEBS Letters

J. Biol. Chem.

Structural analysis of reduced and oxidized Hsp26_S4C

Cloning and purification of Hsp26 and Hsp26_S4C