title: SLLISWD sequence in the 10FNIII domain initiates fibronectin fibrillogenesis.
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.
authors.affiliation: From the Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115.
authors.forename: Elaine P S
authors.initials: EPS
authors.lastname: Gee
authors.affiliation: From the Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115,; the Vascular Biology Program and Departments of Pathology and Surgery, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Deniz
authors.initials: D
authors.lastname: Yüksel
authors.affiliation: the Institute of Medical Engineering and Sciences, Department of Electrical Engineering and Computer Science, and the Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and.
authors.forename: Collin M
authors.initials: CM
authors.lastname: Stultz
authors.affiliation: From the Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115,; the Vascular Biology Program and Departments of Pathology and Surgery, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115,; the Harvard School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138. Electronic address: don.ingber@wyss.harvard.edu.
authors.forename: Donald E
authors.initials: DE
authors.lastname: Ingber
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D058105:Polymerization; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000378:metabolism
chemical_list: D005353:Fibronectins; D010446:Peptide Fragments; D010455:Peptides; C423921:anastellin; D014364:Tryptophan; D005340:Fibrinogen
keywords: Anastellin; Computer Modeling; Cryptic Sites; Extracellular Matrix; Mechanobiology; Peptides; Protein Cross-linking; Protein Self-assembly; RGD