Structure-Function Studies on Small Heat Shock Protein Oligomeric Assembly and Interaction with Unfolded Polypeptides

Abstract

The small heat shock protein (smHSP) and ␣-crystallin genes encode a family of 12-43-kDa proteins which assemble into large multimeric structures, function as chaperones by preventing protein aggregation, and contain a conserved region termed the ␣-crystallin domain. Here we report on the structural and functional characterization of Caenorhabditis elegans HSP16-2, a 16-kDa smHSP produced only under stress conditions. A combination of sedimentation velocity, size exclusion chromatography, and cross-linking analyses on wild-type HSP16-2 and five derivatives demonstrate that the Nterminal domain but not most of the the C-terminal extension which follows the ␣-crystallin domain is essential for the oligomerization of the smHSP into high molecular weight complexes. The N terminus of HSP16-2 is found to be buried within complexes which can accommodate at least an additional 4-kDa of heterologous sequence per subunit. Studies on the interaction of HSP16-2 with fluorescently-labeled and radiolabeled actin and tubulin reveal that this smHSP possesses a high affinity for unfolded intermediates which form early on the aggregation pathway, but has no apparent substrate specificity. Furthermore, both wild-type and C-terminally-truncated HSP16-2 can function as molecular chaperones by suppressing the thermally-induced aggregation of citrate synthase. Taken together, our data on HSP16-2 and a unique 12.6-kDa smHSP we have recently characterized demonstrate that multimerization is a prerequisite for the interaction of smHSPs with unfolded protein as well as for chaperone activity.Molecular chaperones belong to a class of proteins whose function is to interact with and stabilize proteins that are partially or totally unfolded, as is the case when proteins are in the process of being synthesized, translocated across a membrane, or damaged by conditions of cellular stress. Many chaperones are expressed at higher levels during biological stresses, and are members of heat shock protein (HSP) 1 families (1-4). Whereas some chaperones (HSP70, HSP40, and HSP60) are involved in protein folding under normal conditions in vivo (5-7), others such as HSP104 (8 -10), inducible HSP70s (11), and small HSPs (12-15) are known to play important roles in protecting organisms from stress.The small HSPs (smHSPs) form a structurally divergent protein family with members present in Archaea, Bacteria, and Eukarya (16, 17). The presence of an evolutionarily conserved ␣-crystallin domain distinguishes all smHSPs and ␣-crystallins (18 -20). This domain is preceded by an N-terminal domain, which is highly variable in size and sequence, and is followed by a short, poorly conserved C-terminal extension. Some smHSP genes contain an intron which delineates the N-terminal and ␣-crystallin domains (21, 22), and structural studies support a two-domain structure (20,23,24) consisting mostly of ␤-sheets (25, 26) for smHSPs. The C-terminal extensions of smHSPs appear relatively unstructured (27,28) and are known to undergo numerous modifications, including truncati...

Caenorhabditis elegans small heat‐shock proteins Hsp12.2 and Hsp12.3 form tetramers and have no chaperone‐like activity

“…Also for C elegans rec-Hspl6.2, it has been observed that shortening of the N-terminal domain by 15 residues -which gives its Nterminal domain the same length as that of the Hspl2 family -abrogates its ability to form large multimeric complexes [13]. It thus appears that an N-terminal domain of sufficient length is generally required for sHSP multimerization.…”

“…Although the sites of interaction of unfolded substrates with sHSPs are likely to be within or very close to the conserved c~-crystallin domain [7,29], the N-terminal domain has also been implicated in substrate binding [14]. In addition to accessible hydrophobic binding sites, it appears that the presence of flexible polar C-terminal extensions improves the chaperone capacity of sHSPs by enhancing the solubility of the sHSP-substrate complex [13,14]. The loss in the C elegans Hspl2 family of a sufficiently large N-terminal domain, and the absence of a C-terminal tail, would then explain both the inability to form larger multimers and the lack of in vitro chaperone-like activity.…”

Continuous wave and simulated GSM exposure at 1.8 W/kg and 1.8 GHz do not induce hsp16‐1 heat‐shock gene expression in Caenorhabditis elegans

“…This is in contrast to the major hsp70 genes, which are only heat-inducible above 33C [Snutch & Baillie, 1983]. When expressed under stress, the HSP16 proteins form large multimers that act to prevent the aggregation of cellular proteins [Leroux et al, 1997] -and indeed HSP16 co-localises with aggregating proteins in worms [Link et al, 2006]. It has recently been shown that hsp16 genes in general (and hsp16-1 in particular) are key targets for regulation by the DAF-16 FOXO transcription factor in the cell ageing pathway, as well as by HSF-1 in the canonical heat-shock pathway [Hsu et al, 2003;Murphy et al, 2003].…”

Functional Regions of Rice Heat Shock Protein, Oshsp16.9, Required for Conferring Thermotolerance inEscherichia coli

“…Recent studies showed that most LMM HSPs required intact N-terminal domains to form these multimeric complexes (Leroux et al, 1997;Berengian et al, 1999;Lambert et al, 1999). Plant LMM HSPs generally form multimeric protein complexes ranging in size 1 This work was supported by the National Science Council, Republic of China (grant nos.…”

“…Recent studies showed that most LMM HSPs required intact N-terminal domains to form these multimeric complexes (Leroux et al, 1997;Berengian et al, 1999;Lambert et al, 1999). Plant LMM HSPs generally form multimeric protein complexes ranging in size from 200 to 310 kD (Jinn et al, 1995;Lee et al, 1995;Yeh et al, 1995), as shown in rice (Oryza sativa) Oshsp16.9 and pea HSP18.1 consisting of 15 to 18 and 12 subunits, respectively (Jinn et al, 1995;Lee et al, 1995;Yeh et al, 1995).…”