Exploring the roles of substrate-binding surface of the chaperone site in the chaperone activity of trigger factor

Trigger factor (TF) is a key component of the prokaryotic chaperone network, which is involved in many basic cellular processes, such as protein folding, protein trafficking, and ribosome assembly. The major chaperone site of TF has a cradle-like structure in which protein substrate may fold without interference from other proteins. Here, we investigated in vivo and in vitro the roles of hydrophobic and charged patches on the edge and interior of cradle during TF-assisted protein folding. Our results showed that most of the surface of the cradle was involved in TF-assisted protein folding, which was larger than found in early studies. Although the inner surface of cradle was mostly hydrophobic, both hydrophobic and electrostatic patches were indispensable for TF to facilitate correct protein folding. However, hydrophobic patches were more important for the antiaggregation activity of TF. Furthermore, it was found that the patches on the surface of cradle were involved in TF-assisted protein folding in a spatial and temporal order. These results suggest that the folding-favorable interface between the cradle and substrate was dynamic during TF-assisted protein folding, which enabled TF to be involved in the folding of substrate in an aggressive manner rather than acting as a classic holdase.