Molecular dissection of Rab11 binding from coiled-coil formation in the Rab11-FIP2 C-terminal domain

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Rab11-family interacting proteins (Rab11-FIPs) are crucial effectors that bind Rab11 GTPase.
• Rab11 regulates vesicle docking and fusion, essential processes in endosomal trafficking.
• The C-terminal domain of Rab11-FIPs is predicted to form a coiled-coil structure and bind Rab11.

Purpose of the Study:

• To investigate the structural and functional properties of the Rab11-FIP2 C-terminal domain.
• To determine the minimal structural requirements for Rab11 binding.
• To explore the relationship between coiled-coil formation and Rab11 interaction.

Main Methods:

• Limited proteolysis to define the structured core.
• Cross-linking, mutagenesis, and sedimentation equilibrium to analyze dimerization.
• Circular dichroism and isothermal titration calorimetry to assess structure and binding affinity.

Main Results:

• Experimental evidence confirmed coiled-coil formation in Rab11-FIP2's C-terminal domain.
• Rab11-FIP2's coiled-coil domain forms a parallel homodimer in solution.
• Rab11 binding and coiled-coil formation were found to be separable functions, with a C-terminal truncation abolishing binding while retaining structure.

Conclusions:

• Rab11-FIP2's C-terminal domain possesses distinct structural and Rab11-binding functions.
• Dissecting these functions provides insights into the regulation of Rab11-dependent endosomal trafficking.
• This work elucidates a potential mechanism for controlling vesicle transport through Rab11-FIP2 structure-function relationships.