The role of the N-terminal domain in the dynamics of Hsp27 equilibrium dissociation

Abstract

Cells under stress accumulate misfolded and aggregated proteins which can be toxic and may lead to disease states. The induction of small heat shock protein (sHsp) expression enables cells to defend against protein aggregation. Human small heat shock protein 27 (Hsp27) undergoes equilibrium dissociation from an ensemble of large oligomers to a dimer. Equilibrium dissociation, to the dimer, plays an important role in Hsp27 chaperone activity and facilitates high affinity binding to destabilized proteins. In vivo, equilibrium dissociation is regulated by phosphorylation via stress-activated protein kinase MAPKAP kinase 2/3. Hsp27 phosphorylation induces changes in the size and mass distribution of the oligomer ensembles, which modulates Hsp27 chaperone activity. Phosphorylation occurs at multiple serine residues located within the N-terminal domain of Hsp27.

In this work, the structural changes that occur in the N-terminal domain after oligomer dissociation are examined, along with the N-terminal sequence determinants that modulate equilibrium dissociation. The equilibrium between Hsp27 oligomers and dimers was systematically analyzed through cysteine mutagenesis of selected N-terminal residues. The cysteines were derviatized with a sulfhydryl specific spin labels. An advantage of these labels is that they are sensitive to changes in their local environment, in the vicinity of the mutation site. Each spin labeled mutant was analyzed by electron paramagnetic resonance (EPR) for residue environment and solvent accessibility in three different contexts. The first context is in the large ensemble of oligomers (Hsp27-WT), after phosphorylation-induced dissociation, to the dimer, (Hsp27-D3), and the final context after complex formation with model substrate T4 Lysozyme (T4L).

Cysteine mutagenesis identified residues that modulate the Hsp27 dissociation equilibrium. EPR analysis revealed that oligomer dissociation disrupted subunit contacts, subsequently exposing the N-terminal domain to the aqueous environment. After T4L binding, the N-terminal domain transitions from a solvent exposed to a buried environment in the T4L/Hsp27 complex. Furthermore, the EPR data uncovered regions of the N-terminal domain that are highly dynamic. The results support a model of sHsp chaperone activity; in which, unstructured and highly dynamic flexible regions of the N-terminal domain are important for substrate binding.