Tug-Of-War Between Thermodynamic Stability and Actin-Binding Function of Tandem Calponin-Homology Domains

Abstract

Tandem calponin-homology (CH) domains constitute a major class of actin-binding domains that include dystrophin and utrophin, the two key proteins involved in muscular dystrophy. Despite their importance, how their structure controls their function is not understood. Here, we study the contribution of individual CH domains to the actin-binding function of utrophin's tandem CH domain. Co-sedimentation assays indicate that the C-terminal CH2 domain binds weakly to F-actin when compared with the full-length tandem CH domain, consistent with the published results on tandem CH domains. However, the surprise came from the CH1 domain. Isolated CH1 binds strongly to F-actin when compared with the full-length tandem CH domain. These results indicate that CH2 has a negative influence on actin-binding when it is linked with CH1. Thus, the obvious question that arises is why tandem CH domains require CH2, when CH2 is reducing their actin-binding efficiency. To answer, we probed the thermodynamic stabilities of individual CH domains. Isolated CH1 domain is unstable and is prone to serious aggregation. Isolated CH2 is very stable, even more stable than that of the full-length tandem CH domain. This makes utrophin's tandem CH domain as the first example where an isolated domain is more stable than the full-length protein. These results indicate that the main function of CH2 is to stabilize CH1 at the expense of decreasing the actin-binding efficiency. Consistently, the proposed structure of utrophin's tandem CH domain based on earlier X-ray studies indicates a close proximity between the C-terminal helix of CH2 and the N-terminal helix of CH1, and this helix in CH2 becomes more dynamic in the full-length protein when compared with that in the absence of CH1, suggesting a mechanism by which CH2 stabilizes CH1 despite the decrease in actin-binding function.