Ligation of the Fibrin-binding Domain by β-Strand Addition Is Sufficient for Expansion of Soluble Fibronectin

How fibronectin (FN) converts from a compact plasma protein to a fibrillar component of extracellular matrix is not understood. Functional upstream domain (FUD), a polypeptide based on F1 adhesin of Streptococcus pyogenes, binds by anti-parallel beta-strand addition to discontinuous sets of N-terminal FN type I modules, 2-5FNI of the fibrin-binding domain and 8-9FNI of the gelatin-binding domain. Such binding blocks assembly of FN. To learn whether ligation of 2-5FNI, 8-9FNI, or the two sets in combination is important for inhibition, we tested high affinity downstream domain (HADD), which binds by beta-strand addition to the continuous set of FNI modules, 1-5FNI, comprising the fibrin-binding domain. HADD and FUD were similarly active in blocking fibronectin assembly. Binding of HADD or FUD to soluble plasma FN exposed the epitope to monoclonal antibody mAbIII-10 in the tenth FN type III module ((FNIII)-F-10) and caused expansion of FN as assessed by dynamic light scattering. Soluble N-terminal constructs truncated after (FNI)-F-9 or (FNIII)-F-3 competed better than soluble FN for binding of FUD or HADD to adsorbed FN, indicating that interactions involving type III modules more C-terminal than (FNIII)-F-3 limit beta-strand addition to 1-5FNI within intact soluble FN. Preincubation of FN with mAbIII-10 or heparin modestly increased binding to HADD or FUD. Thus, ligation of FNIII modules involved in binding of integrins and glycosaminoglycans, (FNIII)-F-10 and 12-14FNIII, increases accessibility of 1-5FNI. Allosteric loss of constraining interactions among 1-5FNI, (FNIII)-F-10, and 12-14FNIII likely enables assembly of FN into extracellular fibrils.