A mechanism for actin filament severing by malaria parasite actin depolymerizing factor 1 via a low affinity binding...

Area of Science:

• Cell Biology
• Biochemistry
• Structural Biology

Background:

• Actin depolymerizing factor (ADF)/cofilins are crucial for regulating actin turnover in eukaryotic cells.
• These proteins exhibit dual functions: stabilizing actin at high concentrations (decoration) and severing filaments at low concentrations.
• The precise mechanism underlying these contrasting functions of ADF/cofilins has remained unclear.

Purpose of the Study:

• To identify the specific binding interactions of the malaria parasite ADF/cofilin, PfADF1, with actin filaments.
• To elucidate the molecular basis for PfADF1's unique ability to sever actin filaments without stable binding.
• To investigate the potential universality of this novel binding mechanism across different ADF/cofilin proteins.

Main Methods:

• Chemical cross-linking and mass spectrometry (XL-MS) to map protein interactions.
• Structure reconstruction to visualize the binding interface.
• Total internal reflection fluorescence (TIRF) microscopy to observe F-actin severing dynamics.
• Experiments with human cofilin (HsCOF1) and cytochalasin D to probe binding site function.

Main Results:

• A previously unrecognized, low-affinity binding interface on actin filaments targeted by PfADF1 was identified.
• This novel site is distinct from the known 'decoration' binding site.
• TIRF microscopy confirmed this low-affinity site is essential for PfADF1-mediated F-actin severing.
• Blocking the decoration site of human cofilin enhanced its severing activity, suggesting a similar mechanism.

Conclusions:

• PfADF1 utilizes a novel, low-affinity actin-binding site for filament severing, independent of the stable decoration interaction.
• This finding suggests that ADF/cofilins may employ a conserved, secondary binding site for efficient actin filament severing.
• This work provides new insights into the fundamental mechanisms of actin dynamics regulation by ADF/cofilins.