Abstract:
Toxoplasma gondii (TgADF) belongs to a functional subtype characterized by strong G-actin sequestering activity and low F-actin severing activity. Among the characterized ADF/cofilin proteins, TgADF has the shortest length. In order to understand its characteristic properties, we have determined the solution structure of TgADF and studied its backbone dynamics from 15N-relaxation measurements. TgADF has conserved ADF/cofilin fold consisting of a central mixed -sheet comprised of six β-strands which are partially surrounded by three -helices and a C-terminal helical turn. The high G-actin sequestering activity of TgADF is explainable in terms of the highly structurally and dynamically optimized interactions of G-actin with the G-actin binding surface of TgADF. Using ITC, the equilibrium dissociation constant for TgADF and rabbit muscle G-actin, in G-actin buffer, has been directly determined to be 23.81 nM. This reflects the highest affinity determined so far for any ADF/cofilin and G-actin interaction, in the presence of ADP. The F-actin binding site is partially formed, yet it is more rigid than the fully formed F-actin binding site of LdCof. The experimental observations and structural features do not support the interaction of PIP2 with TgADF, and PIP2 does not affect the interaction of TgADF with G-actin. Overall, this study suggests that conformational flexibility of G-actin binding sites enhances the affinity of ADF/cofilin for G-actin, while conformational rigidity of F-actin binding sites of ADF/cofilin is necessary for stable binding to F-actin.
