Structural Stability of Tandem Calponin-Homology (CH) Domains Originates from their C-Terminal CH2 Domain

Abstract

Tandem CH domains form a major class of actin binding domains. In general, the N-terminal CH domain (CH1) weakly binds to F-actin whereas the C-terminal CH domain (CH2) does not bind to actin. However, when CH2 is linked to CH1, the actin binding efficiency increases by more than ten times, which implies a functional cooperativity between the two CH domains. The structural cooperativity underlying this functional cooperativity and the physical mechanism by which CH2 domain enhances the actin-binding efficiency is not understood. In this study, we examined the relative stabilities of the two CH domains of utrophin and dystrophin. The isolated CH1 domain of utrophin does not exist as a stable structure; it is more like a destabilized “molten globule” state. However, its CH2 domain folds to a stable structure, as evident from its alpha-helical spectrum and cooperative melt. Similar to utrophin, the CH2 domain of dystrophin is a well-structured protein, and has similar stability as that of the full-length tandem CH domain. The CH1 domain of dystrophin is quite unstable and aggregates severely. These results indicate that the CH1 domain requires CH2 for its folding, or in other words, CH2 acts as a template for CH1 folding. These stability experiments support the earlier hypothesis proposed based on cryo-EM studies that the major role of CH2 might be to stabilize the tandem CH domain. Additional support comes from the literature: no molecular structures are available for the CH1 domains alone, suggesting that they might be unstable in the absence of CH2. In contrast, structures have been determined for numerous CH2 domains. These experimental observations indicate that the CH2 domain enhances the actin-binding function by imparting structural stability to the tandem CH domains.