Abstract
The small heat shock protein (smHSP) and alpha-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 alpha-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 N-terminal domain but not most of the the C-terminal extension which follows the alpha-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.
Highlights
The small heat shock protein 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
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 small heat shock protein (smHSP) into high molecular weight complexes
The sizes of smHSPs vary considerably, a detailed sequence comparison of known members suggests that the minimal functional unit consists of a core region of about 85 amino acids which is flanked by an N-terminal region of at least 39 residues, and a C-terminal extension of at least 12 residues
Summary
Materials—Porcine heart citrate synthase (CS), bovine serum albumin (BSA), bovine IgG, PIPES, EGTA, and 5-[(4,6-dichlorotriazin-2-. To prepare the vector encoding H6⌬1–15 HSP16-2 (a tagged HSP16-2 lacking the first 15 amino acids), the hsp16-2-containing clone was amplified using polymerase chain reaction and primers. The hsp12.6 coding region was amplified from firststrand cDNA and subcloned into the BglII-HindIII site of a modified pRSET A lacking the polyhistidine-containing NdeI-BamHI fragment [51]. The polyhistidine-containing proteins were purified by Ni2ϩchelate affinity chromatography in 8 M urea as described by the manufacturer (Qiagen), and dialyzed in TEND buffer. Cross-linking of SmHSPs—Cross-linking reactions containing 1.5 M smHSP monomers, 15 M BSA, and 2 mM freshly prepared BS3 were carried out in cross-linking buffer (25 mM MES, pH 7.5, 25 mM NaCl, 0.5 mM DTT) for 30 min at room temperature and analyzed by Western blotting using an antibody against HSP16-2 [50]. Native Gel Analyses of HSP16-21⁄7actin and HSP16-21⁄7tubulin Binary Complexes—The kinetics of HSP16-21⁄7actin and HSP16-21⁄7tubulin binary complex formation were measured by diluting 35S-labeled 7.5 M ureadenatured -actin or -tubulin (prepared as described in Ref. 58) 100fold (final actin and tubulin concentrations were approximately 225 nM) into a solution containing 1.6 M HSP16-2 complex and mixing quickly
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