SLLISWD Sequence in the 10FNIII Domain Initiates Fibronectin Fibrillogenesis

• Published in: The Journal of biological chemistry Vol. 288; no. 29; pp. 21329 - 21340
• Main Authors: Gee, Elaine P.S., Yüksel, Deniz, Stultz, Collin M., Ingber, Donald E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19.07.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Anastellin; Cells, Cultured; Computer Modeling; Cryptic Sites; Extracellular Matrix; Extracellular Matrix - metabolism; Fibrinogen - metabolism; Fibroblasts - metabolism; Fibronectins - chemistry; Fibronectins - metabolism; Glycobiology and Extracellular Matrices; Humans; Hydrophobic and Hydrophilic Interactions; Mechanobiology; Models, Molecular; Molecular Sequence Data; Peptide Fragments - metabolism; Peptides; Peptides - chemistry; Peptides - metabolism; Polymerization; Protein Binding; Protein Cross-linking; Protein Multimerization; Protein Self-assembly; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Unfolding; RGD; Structure-Activity Relationship; Tryptophan - metabolism; Cryptic Sites; Protein Self-assembly; Extracellular Matrix; Peptides; Computer Modeling; Anastellin; Mechanobiology; RGD; Protein Cross-linking
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.462077

Fibronectin (FN) assembly into extracellular matrix is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by cytoskeleton-derived tensional forces transmitted across transmembrane integrins onto RGD binding sequences within the tenth FN type III (10FNIII) domains. These forces unfold 10FNIII to expose cryptic FN assembly sites; however, a specific sequence has not been identified in 10FNIII. Our past steered molecular dynamics simulations modeling 10FNIII unfolding by force at its RGD loop predicted a mechanical intermediate with a solvent-exposed N terminus spanning the A and B β-strands. Here, we experimentally confirm that the predicted 23-residue cryptic peptide 1 (CP1) initiates FN multimerization, which is mediated by interactions with 10FNIII that expose hydrophobic surfaces that support 8-anilino-1-napthalenesulfonic acid binding. Localization of multimerization activity to the C terminus led to the discovery of a minimal 7-amino acid “multimerization sequence” (SLLISWD), which induces polymerization of FN and the clotting protein fibrinogen in addition to enhancing FN fibrillogenesis in fibroblasts. A point mutation at Trp-6 that reduces exposure of hydrophobic sites for 8-anilino-1-napthalenesulfonic acid binding and β-structure formation inhibits FN multimerization and prevents physiological cell-based FN assembly in culture. We propose a model for cell-mediated fibrillogenesis whereby cell traction force initiates a cascade of intermolecular exchange starting with the unfolding of 10FNIII to expose the multimerization sequence, which interacts with strand B of another 10FNIII domain via a Trp-mediated β-strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly. Background: Fibronectin matrix assembly is mediated by cell traction at the RGD loop of 10FNIII, which is predicted to unravel β-strands A and B. Results: Sequence SLLISWD from strand B initiates fibronectin multimerization. Conclusion: SLLISWD mediates cell-mediated fibronectin fibril assembly. Significance: This matrix motif provides insight into physiological fibronectin fibrillogenesis with utility in initiating matrix assembly for tissue repair.