Computational Analysis of the Interaction Between Paired CH-Domains - Implications for their F-actin Bound Conformations

Paired calponin-homology (CH)-domains form the actin-binding domains (ABD) of an important family of cytoskeletal proteins, including utrophin, dystrophin, fimbrin, alpha-actinin, plectin, and spectrin. While the crystal structures of various ABDs exhibit extensive inter-CH interactions and display a compact conformation, the functional conformation of F-actin-bound ABDs is still unresolved. Studies suggest that upon binding to F-actin the compact conformation observed in crystal structures persists; others suggest that the CHs separate and the ABD becomes extended. To resolve this, we calculated the energy of inter-CH interfaces by computational alanine scanning (ΔΔGbinding = 32.9kcal/mol) and computed the energy (6.7kcal/mol) of the utrophin-F-actin interaction based on the 13uM Kd (Moores, 2000). This sets a lower limit for the ΔΔGbinding for the open CH model of the utrophin-F-actin interaction at 39.6kcal/mol. Without ABD-actin crystal structures, we cannot compare the interfaces to see if there is enough energy to bind the ABD while disassociating the CH domains. Instead, we computed the minimum energy density of a utrophin-F-actin interface necessary to open the CH domains and compared this value with other known actin interfaces. We divided the area (610A2 obtained from the closed fimbrin-fitted model [Galkin 2008]) by the core binding energy (39.6kcal/mol) to get 15.6A2/kcal/mol; an energy density that is statistically significantly different (p=0.0491) than the mean value for other actin-binding proteins (26.7A2/kcals/mol), including DNase-I, gelsolin, profilin, and DBP. This seems to contraindicate a model of an ABD extended into solution because the actin interface proposed to disassociate the CH domains would be unusually energy dense with respect to other known actin binding interfaces and if this solvent exposed model is an intermediate on the path to extended ABD binding, this would preclude the formation of an extended interface on actin.