STRUCTURE-FUNCTION STUDIES ON SMALL HEAT-SHOCK-PROTEIN OLIGOMERIC ASSEMBLY AND INTERACTION WITH UNFOLDED POLYPEPTIDES

The small heat shock protein (smHSP) and alpha-crystallin genes encode a family of 12-43-kDa proteins which assemble into large multimeric s tructures, function as chaperones by preventing protein aggregation, a nd contain a conserved region termed the alpha-crystallin domain, Here we report on the structural and functional characterization of Caenor habditis elegans HSP16-2, a 16-kDa smRSP produced only under stress co nditions. A combination of sedimentation velocity, size exclusion chro matography, and cross-linking analyses on wild-type HSP16-2 and five d erivatives 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 wei ght complexes. The N terminus of HSP16-2 is found to be buried within complexes which can accommodate at least an additional 4-kDa of hetero logous sequence per subunit. Studies on the interaction of HSP16-2 wit h 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 HS P16-2 can function as molecular chaperones by suppressing the thermall y-induced aggregation of citrate synthase., Taken together our data on HSP16-2 and a unique 12.6-kDa smHSP we have recently characterized de monstrate that multimerization is a prerequisite for the interaction o f smHSPs with unfolded protein as well as for chaperone activity.