C-terminal β-strand swapping in a consensus-derived fibronectin Type III scaffold

Area of Science:

• Protein structure and biophysics
• Molecular biology
• Structural biology

Background:

• Fibronectin Type III domains are ubiquitous in cell adhesion and signaling.
• Understanding protein aggregation is crucial for disease and biotechnology.
• Tencon domains are a specific subclass of fibronectin Type III domains.

Purpose of the Study:

• To determine the crystal structures of six Tencon domain variants.
• To elucidate the structural mechanisms underlying Tencon domain aggregation.
• To correlate structural findings with solution-based aggregation properties.

Main Methods:

• X-ray crystallography to determine high-resolution structures.
• Size Exclusion Chromatography (SEC) to assess solution aggregation.
• Sequence analysis of FG loop variations.

Main Results:

• Five of six Tencon variants formed 3D domain-swapped dimers.
• Domain swapping involved the C-terminal β-strand, blocking the target-binding surface.
• FG loop sequence, particularly insertions and non-glycine residues at position 77, influences swapping and oligomerization.
• Longer FG loops promoted hexameric or helical oligomers, correlating with solution aggregation.

Conclusions:

• 3D domain swapping is a key mechanism for Tencon domain aggregation.
• FG loop sequence and length are critical determinants of dimerization and oligomerization.
• Structural insights can guide the engineering of Tencon domains with tailored biophysical properties.