Module-module interactions in the cell binding region of fibronectin: stability, flexibility and specificity

Area of Science:

• Structural Biology
• Protein Engineering
• Biophysics

Background:

• Mosaic proteins are composed of multiple modules whose interactions dictate overall structure and function.
• The RGD-dependent cell binding region of human fibronectin, comprising fibronectin type III modules 9 and 10, is crucial for cell adhesion.

Purpose of the Study:

• To investigate the structural significance of module-module interactions in the fibronectin type III 9-10 pair.
• To establish a relationship between intermodular protein-protein interactions and the structural properties of the module pair.

Main Methods:

• Protein engineering of single and double module constructs, including variations with poly(glycine) linkers.
• Thermodynamic analysis using equilibrium and kinetic unfolding experiments.
• Nuclear Magnetic Resonance (NMR) spectroscopy, including chemical shift analysis and 15N relaxation studies.

Main Results:

• Non-specific protein-protein interactions contribute significantly to the thermodynamic stabilization and motional constraint of the fibronectin type III 9-10 module pair.
• Specific interactions are observed only in the wild-type construct and diminish rapidly with increased linker length.
• Despite low specificity, these interactions precisely orient the modules and restore full biological activity.

Conclusions:

• Individual modules in mosaic proteins can attain substantial motional constraint and mutual stabilization via non-specific interactions.
• Intricate and specific interfaces are not always required for stabilizing module-module interactions in complex proteins.
• This finding has implications for understanding protein structure-function relationships and designing novel mosaic proteins.