Abstract
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.
Highlights
Fibronectin matrix assembly is mediated by cell traction at the RGD loop of 10FNIII, which is predicted to unravel -strands A and B
Unfolded N-terminal Sequence of Predicted 10FNIII Intermediate Initiates FN Multimerization—Our past SMD simulations modeling the unfolding pathway of 10FNIII under tension between its N terminus and the physiological integrin-binding RGD loop predicted an unfolded intermediate with an unraveled N terminus through the second -strand [26] (Fig. 1B). Because this kinetic intermediate is sampled for extended simulation times while under tension, we proposed that such a partially unfolded structure might expose cryptic assembly sites that trigger FN assembly
A 23-amino acid peptide mimic designed from this unfolded terminus is shown to be a cryptic sequence in 10FNIII that initiates FN multimerization as its scrambled sequence, the unexposed -strand E, and natively folded 10FNIII are unable to initiate similar multimerization results
Summary
Fibronectin matrix assembly is mediated by cell traction at the RGD loop of 10FNIII, which is predicted to unravel -strands A and B. 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; a specific sequence has not been identified in 10FNIII. The RGD cell-binding sequence and neighboring synergy site are required for FN assembly by the ␣51 integrin [14, 22], how 10FNIII transduces force applied at its RGD loop to initiate fiber assembly remains unclear Both single molecule atomic force microscopy experiments and steered molecular dynamics (SMD) simulations suggest that 10FNIII is one of the mechanically weakest FNIII domains [19, 23]. We set out to test whether this predicted exposed region contributes cryptic assembly sites and to identify the minimal peptide sequence within the 10FNIII domain that is sufficient to induce FN self-assembly
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