Semi-empirical structure determination of Escherichia coli Hsp33 and identification of dynamic regulatory elements for the activation process

Abstract

The activation process of the redox-regulated chaperone heat shock protein 33 (Hsp33) is constituted by the oxidation-induced unfolding of the C-terminal zinc-binding domain and concomitant oligomerization of the N-terminal core domain. Herein, the semi-empirical solution structure of Escherichia coli Hsp33 in the reduced, inactive form was generated through conformational space annealing calculations, utilizing minimalistic NMR data and multiple homology restraints. The various conformations of oxidized Hsp33 and some mutant forms were also investigated in solution. Interestingly, a specific region concentrated around the interdomain linker stretch and its interacting counterparts, the N-terminal β-strand 1 and α-helix 1, hardly showed up as signals in the NMR measurements. The NMR spectra of an Hsp33 derivative with a six-residue deletion in the disordered N-terminus implied a plausible conformational exchange associated with the identified region, and the corresponding exchange rate appeared slower than that of the wild type. Subsequent mutations that destroyed the structure of the β1 or α1 elements resulted in the formation of a reduced but active monomer, without the unfolding of the zinc-binding domain. Collectively, structural insights into the inactive and active conformations, including wild-type and mutant proteins, suggest that the dynamic interactions of the N-terminal segments with their contacting counterpart, the interdomain linker stretch, in the reduced, inactive state are the structural determinants regulating the activation process of the post-translationally regulated chaperone, Hsp33.