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Hassane S. Mchaourab, Sanjay Mishra, Hanane A. Koteiche, Jian Shi, Dewight Williams, Phoebe L. Stewart; Role of the N-terminal Domain in Polydispersity and Substrate Binding by Small Heat Shock Proteins: Lessons from Hsp16.5. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1612.
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Lens α-crystallin and small heat shock proteins have a highly divergent N-terminal region. We have previously shown that insertion of a 14 amino acid peptide from human Hsp27 at the junction of the N-terminal region and the α-crystallin domain of Methanocaldococcus jannaschii Hsp16.5 induces the formation of a highly symmetric oligomer with twice the number of subunits compared to WT. In this work, we explore the mechanism by which sequence divergence in the N-terminal region defines oligomer symmetry and order and is critical for substrate binding
We systematically varied the length and sequence of the Hsp27 peptide insertion. The resulting Hsp16.5 variants were then analyzed by electron microscopy, light scattering and blue native gel electrophoresis. Furthermore, the structure of the R107G mutant, an equivalent mutation to αB-crystallin R120G associated with cardiomyopathy, was determined by cryoEM.
The data demonstrate that insertions at multiple positions in the N-terminal region of Hsp16.5 induce the foramtion of larger assemblies than the WT. Both negative stain EM and blue native gel electrophoresis reveal the presence of a heterogeneous, loosely packed oligomer with an average of 36 subunits. This intermediate is populated in response to insertions as short as 6 amino acids. In contrast, the transition to the symmetric 48-subunit assembly requires specific sequence elements and a minimum peptide length specifically isnerted at the junction between the N-terminal and α-crystallin domain regions of Hsp16.5. The structure of the R107G mutant shows that the most significant conformational changes occur in the N-terminal region within the core of the oligomer. This mutant has higher binding affinity to destabilized mutants of T4 lysozyme relative to the WT consistent with a critical role of the N-terminal region in substrate binding.
The uncovered plasticity of the ordered Hsp16.5 demonstrates the flexibility in packing of α-crystallin domain dimers that generates the spectrum of quaternary structures observed for the small heat shock protein superfamily.
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