Heparin binding by fibronectin module III-13 involves six discontinuous basic residues brought together to form a...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Fibronectin's thirteenth type III domain (III-13) binds heparin, containing a proposed heparin-binding consensus sequence (Arg6-Arg7-Ala8-Arg9).
• Previous studies showed conflicting results regarding the affinity of this consensus sequence for heparin.

Purpose of the Study:

• To investigate the structural basis of heparin binding by fibronectin type III-13.
• To determine the contribution of the Arg6-Arg7-Ala8-Arg9 motif and other residues to heparin affinity.

Main Methods:

• Generated a 3D model of fibronectin type III-13.
• Expressed wild-type and mutant (single-site substitutions) III-13 domains in E. coli.
• Assessed heparin-binding affinity using fluorescence anisotropy with fluorescein-labeled heparin.

Main Results:

• The Arg6-Arg7-Ala8-Arg9 sequence alone has low heparin affinity.
• A cluster of six positively charged residues, including Arg6, Arg7, Arg9, Arg23, Lys25, and Arg54, forms a "cationic cradle" for heparin binding.
• Mutations in these residues significantly reduced heparin binding affinity, with R54S showing a 3-fold decrease and others a >10-fold decrease.

Conclusions:

• The heparin-binding site in fibronectin type III-13 is more complex than a simple consensus sequence.
• Multiple charged residues remote in sequence are brought together in the folded domain to create the functional binding site.
• This "cationic cradle" structure explains the high affinity of fibronectin III-13 for heparin.