Solution structure and dynamics of ADF from Toxoplasma gondii

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Toxoplasma gondii ADF (TgADF) is a functional ADF/cofilin subtype with high G-actin sequestering and low F-actin severing activity.
• TgADF is notably short and lacks a C-terminal helix crucial for F-actin binding in other ADF/cofilin proteins.

Purpose of the Study:

• To elucidate the structural and dynamic basis of TgADF's unique actin-binding properties.
• To understand the molecular interactions governing TgADF's strong G-actin affinity and weak F-actin interaction.

Main Methods:

• Solution structure determination of TgADF using NMR spectroscopy.
• ¹⁵N-relaxation measurements to study backbone dynamics.
• Isothermal Titration Calorimetry (ITC) to quantify G-actin binding affinity.

Main Results:

• TgADF exhibits a conserved ADF/cofilin fold with a flexible G-actin binding site and a rigid, partially formed F-actin binding site.
• High affinity for G-actin (Kd = 23.81 nM) is attributed to optimized interactions.
• No interaction with PIP2 was observed, and PIP2 did not influence TgADF-G-actin binding.

Conclusions:

• Conformational flexibility in the G-actin binding site enhances TgADF's affinity for G-actin.
• Conformational rigidity in F-actin binding sites is essential for stable F-actin binding in conventional ADF/cofilins.
• TgADF's structural and dynamic features explain its distinct functional specialization.