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Mammalian small heat shock proteins (sHSP) form polydisperse and dynamic oligomers that undergo equilibrium subunit exchange. Current models of their chaperone activity hypothesize that recognition and binding of protein non-native states involve changes in the oligomeric state. The equivalent thermodynamic representation is a set of three coupled equilibria that includes the sHSP oligomeric equilibrium, the substrate folding equilibrium, and the equilibrium binding between the sHSP and the substrate non-native states. To test this hypothesis and define the binding-competent oligomeric state of human Hsp27, we have perturbed the two former equilibria and quantitatively determined the consequences on binding. The substrate is a set of T4 lysozyme (T4L) mutants that bind under conditions that favor the folded state over the unfolded state by 10 2 -10 4 -fold. The concentration-dependent oligomer equilibrium of Hsp27 was perturbed by mutations that alter the relative stability of two major oligomeric states including phosphorylation-mimicking mutations that result in the dissociation to a small multimer over a wide range of concentrations. Correlation of binding isotherms with size exclusion chromatography analysis of the Hsp27 oligomer equilibrium demonstrates that the multimer is the binding-competent state. Binding occurs through two modes, each characterized by different affinity and number of binding sites, and results in T4L⅐Hsp27 complexes of different hydrodynamic properties. Mutants of the Hsp27 phosphorylation mimic that reverse the reduction in oligomer size also reduce the extent of T4L binding. Taken together, these results suggest a central role for the oligomeric equilibrium in regulating the chaperone activity of sHSP. The mutants identify sequence features important for modulating this equilibrium.

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Human small heat shock protein 27 (Hsp27) undergoes concentration-dependent equilibrium dissociation from an ensemble of large oligomers to a dimer. This phenomenon plays a critical role in Hsp27 chaperone activity in vitro enabling high affinity binding to destabilized proteins. In vivo dissociation, which is regulated by phosphorylation, controls Hsp27 role in signaling pathways. In this study, we explore the sequence determinants of Hsp27 dissociation and define the structural basis underlying the increased affinity of Hsp27 dimers to client proteins. A systematic cysteine mutagenesis is carried out to identify residues in the N-terminal domain important for the equilibrium between Hsp27 oligomers and dimers. In addition, spin labels were attached to the cysteine mutants to enable electron paramagnetic resonance (EPR) analysis of residue environment and solvent accessibility in the context of the large oligomers, upon dissociation to the dimer, and following complex formation with the model substrate T4 Lysozyme (T4L). The mutagenic analysis identifies residues that modulate the equilibrium dissociation in favor of the dimer. EPR analysis reveals that oligomer dissociation disrupts subunit contacts leading to the exposure of Hsp27 N-terminal domain to the aqueous solvent. Moreover, regions of this domain are highly dynamic with no evidence of a packed core. Interaction between T4L and sequences in this domain is inferred from transition of spin labels to a buried environment in the substrate/Hsp27 complex. Together, the data provides the first structural analysis of sHSP dissociation and supports a model of chaperone activity wherein unstructured and highly flexible regions in the N-terminal domain are critical for substrate binding.

We have identified sequence and structural determinants of oligomer size, symmetry, and polydispersity in the small heat shock protein superfamily. Using an insertion mutagenesis strategy that mimics evolutionary sequence divergence, we induced the ordered oligomer of Methanococcus jannaschii Hsp16.5 to transition to either expanded symmetric or polydisperse assemblies. A hybrid approach combining spin labeling EPR and cryoelectron microscopy imaging at 10 Å resolution reveals that the underlying plasticity is mediated by a packing interface with minimal contacts and a flexible C-terminal tether between dimers. Twenty-four dimeric building blocks related by octahedral symmetry assemble into the expanded symmetric oligomer. In contrast, the polydisperse variant has an ordered dimeric building block that heterogeneously packs to yield oligomers of various sizes. Increased exposure of the N-terminal region in the Hsp16.5 variants correlates with enhanced binding to destabilized mutants of T4 lysozyme, whereas deletion of this region reduces binding. Transition to larger intermediates with enhanced substrate binding capacity has been observed in other small heat shock proteins including lens ␣-crystallin mutants linked to congenital cataract. Together, these results provide a mechanistic perspective on substrate recognition and binding by the small heat shock protein superfamily.In the crowded molecular environment of the cell, five classes of heat shock proteins (HSP) 4 act as molecular chaperones by suppressing protein aggregation through the selective binding of proteins in non-native states (1, 2). Subsequent processing of the bound polypeptides involves either release and refolding or delivery to degradation machineries. Small heat shock proteins (sHSP) are members of a superfamily of oligomeric proteins characterized by a conserved module in the C-terminal region of their sequences referred to as the ␣-crystallin domain (3, 4). In vitro, sHSP are protein stability sensors that can differentially bind mutants with similar structures in the folded state but different free energies of unfolding (5, 6). Substrates are bound with efficiencies that can reach 1 substrate molecule/subunit of sHSP and without direct input of ATP energy (7-9). Bound substrates are protected against heat inactivation and can be released from sHSP complexes by other members of the chaperone network (7, 10). sHSP play critical roles in maintaining vertebrate lens transparency (11) and in a number of physiologic processes (12)(13)(14). Their expression in Caenorhabditis elegans promotes longevity and delays the onset of polyglutamine protein aggregation (15).The structural basis of substrate recognition and binding by sHSP continues to be enigmatic. Most sHSP assemble into polydisperse and dynamic oligomers, and their heterogeneity is enhanced by substrate binding. Available high resolution structures are for three ordered, symmetrical sHSP assemblies, Hsp16.5 from Methanococcus jannaschii (MJ) (16), Hsp16.9 from wheat (17), and the more ...