HspB5 Chaperone Structure and Activity Are Modulated by Chemical-Scale Interactions in the ACD Dimer Interface

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Folding

Background:

• Small heat shock proteins (sHsps) are ATP-independent molecular chaperones.
• They possess a conserved alpha-crystallin domain (ACD) and prevent protein aggregation.
• Their dynamic structures and substrate interactions are not fully understood.

Purpose of the Study:

• Investigate the impact of disease-associated mutations in the HspB5 ACD on chaperone activity.
• Elucidate the physicochemical properties influencing sHsp structure and function.
• Understand the substrate selectivity of sHsps.

Main Methods:

• In vitro chaperone activity assays using disease-relevant HspB5 mutations (D109A, F113Y, R116C, R120G, R120C).
• Structural analysis using ANS fluorescence and Circular Dichroism (CD) spectroscopy.
• Light-scattering assays to evaluate substrate-sHsp interactions.

Main Results:

• Mutation Y113F maintained efficient holdase activity.
• Mutations D109A and R120G significantly reduced holdase activity, impacting substrate interactions.
• Chaperone activity reduction varied among mutants and was substrate-specific, indicating selective interactions.

Conclusions:

• Key electrostatic interactions within the sHsp dimer are crucial for structural stability.
• These interactions influence higher-order sHsp assembly and substrate binding.
• sHsps exhibit substrate selectivity, mediated by specific interactions defining their holdase activity.