MFG-E8: a model of multiple binding modes associated with ps-binding proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Membrane-binding proteins typically exhibit a two-state binding model (bound/unbound).
• A single binding interface can display multiple binding modes, but these are difficult to distinguish.
• Milk fat globule EGF factor 8 (MFG-E8) binds phosphatidylserine (PS), a marker for cellular apoptosis and malignancy.

Purpose of the Study:

• To investigate the binding modes of MFG-E8 to lipid membranes.
• To elucidate the factors influencing MFG-E8's binding affinity and cooperativity.
• To develop a new model for analyzing multi-modal protein-lipid interactions.

Main Methods:

• Utilized a model system of lipid vesicles with varying phosphatidylserine (PS) mole fractions.
• Employed tryptophan fluorescence spectral shift assays to measure binding affinity.
• Applied X-ray reflectivity experiments to assess membrane packing effects.

Main Results:

• Identified three distinct binding modes for MFG-E8, each correlating with a specific number of PS lipids.
• Demonstrated that the availability of negatively charged lipids enhances MFG-E8's binding cooperativity.
• Found that membrane packing influences MFG-E8's binding modes.

Conclusions:

• MFG-E8 exhibits complex, multi-modal binding to lipid membranes, not a simple two-state interaction.
• Electrostatic interactions with PS lipids and membrane packing are key determinants of MFG-E8 binding.
• The developed methods can be applied to study other membrane-binding proteins and their interactions with membrane attributes.