Mutants in a small heat shock proteins that affect the oligomeric state: analysis and allele specific suppression.

[Advanced Search][Browse the Archive] Institution: UNIV OF MASSACHUSETTS, Amherst Sign In Advertisement 10,000 Expression ORF Clones for $98 each Advertisement JBC\u27s Young Investigator Award: Learn More Mutants in a Small Heat Shock Protein That Affect the Oligomeric State ANALYSIS AND ALLELE-SPECIFIC SUPPRESSION* Kim C. Giese and Elizabeth Vierling‡ + Author Affiliations Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721 ‡ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biophysics, University of Arizona, 1007 E. Lowell St., Tucson, AZ 85721. Tel.: 520-621-1601; Fax: 520-621-3709; E-mail: vierling@email.arizona.edu. Next Section Abstract Oligomerization is an essential property of small heat shock proteins (sHSPs) that appears to regulate their chaperone activity. We have examined the role of conserved hydrophobic residues that are postulated to stabilize sHSP oligomers. We identified a mutation of Synechocystis Hsp16.6 that impairs function in vivo and in vitro. The V143A mutation is in the C-terminal extension, a region predicted to form an oligomeric interaction with a hydrophobic region that includes the site of a previously characterized mutation, L66A. Both mutants were dimeric, but V143A had a stronger oligomerization defect than L66A. However, V143A protected a model substrate better than L66A. This suggests that although the two regions both play a role in oligomerization, they are not equivalent. Nevertheless, the addition of either dimeric sHSP enhanced the in vitro chaperone activity of wild type Hsp16.6, consistent with models that the sHSP dimers initiate interactions with substrates. Suppressor analysis of V143A identified mutations in the N terminus that restored activity by restabilizing the oligomer. These mutants were allele-specific and unable to suppress L66A, although they suppressed a dimeric C-terminal truncation of Hsp16.6. Conversely, suppressors of L66A were unable to suppress either V143A or the truncation, although they, like suppressors of V143A, stabilize the Hsp16.6 oligomer. We interpret these data as evidence that the mutations V143A and L66A stabilize two different dimeric structures and as further support that sHSP dimers are active species. Previous SectionNext Section Small heat shock proteins (sHSPs)1 are a family of ATP-independent chaperones that, in vitro, can protect denaturing proteins from irreversible aggregation by forming large, soluble complexes with their substrates (for reviews, see Refs. 1-3). sHSPs do not refold substrates, but sHSP-protected substrates can be refolded by ATP-dependent chaperones such as Hsp70/DnaK (4, 5). Although the role of sHSPs in vivo is not well defined, established in vitro assays for sHSP activity (i.e. protection of model substrates from aggregation and their subsequent refolding by ATP-dependent chaperones) appear to be closely related to sHSP functions in vivo. In Synechocystis sp. strain PCC 6803 (Synechocystis), a missense mutation of the single sHSP in this organism, Hsp16.6, causes loss of both protection of a model substrate in vitro and the ability of cells to survive heat treatment (thermotolerance). Both activities are restored by intragenic suppressors of this mutant (6). Mogk et al. (7) demonstrated that the sHSPs in Escherichia coli act with both DnaK and ClpB to facilitate the removal of aggregated protein after heat stress, in good agreement with in vitro observations of Hsp70/DnaK-dependent reactivation of sHSP-protected substrates (8, 9). Thus, it appears that sHSPs function to prevent or reduce irreversible protein aggregation during heat stress in vivo. The mechanism of the chaperone activity of sHSPs is unknown. Current models suggest that changes in sHSP oligomerization are essential. Almost all sHSPs form oligomers (2), and these can be destabilized at high temperatures (10, 11). Disassembly of the oligomer into dimers has been proposed to be a required step in the chaperone process (10). Disassembly could produce an active state of the sHSP that can bind unfolding proteins (10), perhaps by exposure of hydrophobic residues that bind substrates (5, 11, 12). However, the oligomeric state is also essential, as it appears that reassembly of sHSP dimers into a complex with substrate is required for protection of substrates (6, 13-16). Therefore, identification of contacts involved in oligomerization is important for understanding the mechanism of sHSP chaperone activity. The crystal structures of two sHSPs provide information about regions involved in oligomerization. The structures of Methanococcus jannaschii Hsp16.5 (17) and Triticum aestivum Hsp16.9 from wheat (11) reveal that despite the difference in the size of their oligomers (24 and 12 subunits, respectively), there is a high degree of similarity between the two sHSPs. Both proteins form oligomers that are composed of dimers, and the conserved ∼100 amino acid α-crystallin domains (18) have very similar β-sandwich structures. Oligomerization of M. jannaschii Hsp16.5 and T. aestivum Hsp16.9 involves a conserved interdimer interface between two hydrophobic regions: a “patch” of hydrophobicity on one surface of the β-sandwich of the α-crystallin domain and the hydrophobic residues in a basic-X(I/V)X(I/V) motif of the C-terminal extension (β-strand 10). The conservation of these residues among sHSPs (18) is consistent with essential functions of these regions. The hypothesis that interactions between the C-terminal extension and the hydrophobic patch are essential to sHSP oligomerization and function is supported by some, but not all, mutagenesis experiments in vitro and has not been tested in vivo. Mutation of either Ile residue to Ala in the basic-X(I/V)X(I/V) motif of HspH from Bradyrhizobium japonicum destabilized the oligomer and led to loss of protection of citrate synthase from aggregation (15). A truncation of the C terminus that deleted this motif also reduced the oligomerization of M. jannaschii Hsp16.5 (19) but not of Hsp16.2 from Caenorhabditis elegans (13). Furthermore, while Kim et al. (19) found that the C-terminal truncation of M. jannaschii Hsp16.5 led to formation of a tetramer, it was still able to maintain substrate solubility. The role of the hydrophobic patch is even less well examined. We identified a mutation of a conserved hydrophobic residue in the patch, L66A of Synechocystis Hsp16.6, that severely reduced both oligomeric stability and chaperone activity (6), but this region has not been investigated in other sHSPs. Although on the whole, these studies argue for a role of the hydrophobic C-terminal motif and patch in both oligomerization and chaperone activity, the requirements for either are not certain. Furthermore, these studies have primarily assayed sHSP activity using model substrates in vitro, making it possible that the results do not reflect cellular needs for sHSP activity. We took advantage of the requirement of Hsp16.6 for thermotolerance in Synechocystis to dissect genetically the role of the C-terminal motif and to compare it with that of the hydrophobic patch. The relationship of in vivo phenotypes to oligomeric stability and chaperone activity was also compared. Point mutants at Val143, the second hydrophobic residue in the basic-X(I/V)X(I/V) motif of Hsp16.6, decreased the viability of heat-stressed cells, providing the first direct evidence for the importance of this motif in vivo. The V143A protein was dimeric and had impaired chaperone activity in vitro. V143A disrupted oligomeric stability more, but chaperone activity less, than L66A, a mutation in the hydrophobic patch, suggesting the two hydrophobic regions do not have equivalent functions. Intragenic suppressors of V143A were then selected in Synechocystis as second-site mutations that restored cellular thermotolerance. These suppressors were located in the N terminus and greatly stabilized the Hsp16.6 oligomer. Contrary to expectations, the suppressors of V143A were not able to suppress L66A. Likewise, suppressors of L66A, which also stabilize the Hsp16.6 oligomer (6), did not suppress V143A. In total, although both V143A and L66A form stable dimers, there are structural and/or functional differences between the two mutants. We suggest that they may stabilize two distinct functional states of Hsp16.6