Structural And Mechanistic Insights Into Regulation Of Chaperone Activity In Sulfolobus Solfataricus Hsp14.1 And Hsp20.1

Small heat shock proteins(s Hsps) are a superfamily of molecular chaperones existing in all living creatures and multiple biological tissues. Under stressed conditions, s Hsps play essential roles through binding to denatured client proteins, preventing them from aggregating and precipitating. Loss of s Hsp function sensitizes organisms to environment stresses such as heating, hypoxia, acid/alkaline, etc. In humans, malfunction of s Hsps results in diseases such as neurodegenerative disorder, cataract and autoimmune diseases, etc. However, how s Hsps bind clients in stress conditions and how they enable the cells to tolerate stresses are poorly understood. Accumulating data indicate that the N-terminal domain(NTD) of s Hsps consisting of ~25 residues is responsible for binding to client proteins, but the binding mechanism and regulation of the chaperone activity remain elusive.It remains unclear how the NTD adapts to different kinds of denatured client proteins that are diverse in size, shape, surface hydrophobicity and charge. Thus far, there has not been a structure of s Hsp that provides a clear, high-resolution view of the NTD in the active state. Since denatured proteins are heterogeneous without defined three-dimensional structure, it is difficult to directly study the structure of the complex formed by s Hsps and non-native clients.In this study, we combined in vitro and in vivo functional studies with structural investigation to provide the molecular mechanism of how NTD of a small heat shock protein 14.1 from Sulfolobus solfatataricus P2(Ss Hsp14.1) changes its conformation and adapts to nonnative clients.We determined the crystal structures of the wild-type s Hsp14.1, mutants A102 D and Del-C4. All three structures reveal well-defined NTD, but the NTD conformations are remarkably different. The NTDs of the wild-type s Hsp dimer are straight helices, but are severely kinked and broken in the mutants. In the context of the dimer, the kinked and broken NTDs lead to formation of a hydrophobic pocket compatible with recognizing unfolded client proteins. Our functional mutagenesis data indeed show that mutating key hydrophobic residues in this pocket drastically reduced the bacteria’s viability upon heat treatment. In contrast, the straight NTD helices in the wild-type dimer do not constitute a defined hydrophobic pocket. The structural difference in NTDs between the wild-type and mutants thus suggests a molecular switch that turns on the chaperone activity of s Hsp in the resting state. The same mechanism may apply to other similar s Hsps in the family.Based on the structural data, it is certainly agreed that two dimerization models depend on the presence or absence of a β6 strand to differentiate nonmetazoan s HSPs from metazoan s HSPs. Here, we report the Sulfolobus solfataricus Hsp20.1 ACD dimer structure, which shows a distinct dimeric interface. We observed that, in the absence of β6, Hsp20.1 dimer does not depend on β7 strand for forming dimer interface as metazoan s HSPs, nor dissociates to monomers. This is in contrast to other published s HSPs. Our structure reveals a variable, highly polar dimer interface that has advantages for rapid subunits exchange and substrate binding. Remarkably, we find that the C-terminal truncation variant has chaperone activity comparable to that of wild-type despite lack of the oligomer structure. Our further study indicates that the N-terminal region is essential for the oligomer and dimer binding to the target protein.A great number of studies have proven that s Hsps protect cells by inhibiting protein aggregation under heat stress, while little is known about their function to protect cells under acid stress. In this work, we show that Hsp20.1 and Hsp14.1 oligomers dissociated to smaller oligomeric species or even dimer/monomer at low p H(p H 4.0 and p H 2.0), whereas no prominent quaternary structural changes were seen at 50 °C. Both oligomers and smaller oligomeric species exhibited abilities to suppress clients aggregation at low p H and at 50 °C. These results suggest that s Hsps may function in different modes in different stressed conditions.