Increased surface charge in the protein chaperone Spy enhances its anti-aggregation activity

• Published in: The Journal of biological chemistry 2020-10, Vol.295 (42), p.14488-14500
• Main Authors: He, Wei, Zhang, Jiayin, Sachsenhauser, Veronika, Wang, Lili, Bardwell, James C.A., Quan, Shu
• Format: Article
• Language: English
• Subjects: Animals; Cattle; chaperone-substrate interaction; conformational change; electrostatic interaction; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Hydrophobic and Hydrophilic Interactions; hydrophobic interaction; Kinetics; Lactalbumin - chemistry; Lactalbumin - metabolism; molecular chaperone; Mutagenesis, Site-Directed; Periplasmic Proteins - chemistry; Periplasmic Proteins - genetics; Periplasmic Proteins - metabolism; Protein Aggregates; protein aggregation; Protein Binding; protein engineering; protein folding; Protein Structure and Folding; Spy; Static Electricity; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.012300

Chaperones are essential components of the protein homeostasis network. There is a growing interest in optimizing chaperone function, but exactly how to achieve this aim is unclear. Here, using a model chaperone, the bacterial protein Spy, we demonstrate that substitutions that alter the electrostatic potential of Spy's concave, client-binding surface enhance Spy's anti-aggregation activity. We show that this strategy is more efficient than one that enhances the hydrophobicity of Spy's surface. Our findings thus challenge the traditional notion that hydrophobic interactions are the major driving forces that guide chaperone–substrate binding. Kinetic data revealed that both charge- and hydrophobicity-enhanced Spy variants release clients more slowly, resulting in a greater “holdase” activity. However, increasing short-range hydrophobic interactions deleteriously affected Spy's ability to capture substrates, thus reducing its in vitro chaperone activity toward fast-aggregating substrates. Our strategy in chaperone surface engineering therefore sought to fine-tune the different molecular forces involved in chaperone–substrate interactions rather than focusing on enhancing hydrophobic interactions. These results improve our understanding of the mechanistic basis of chaperone–client interactions and illustrate how protein surface–based mutational strategies can facilitate the rational improvement of molecular chaperones.