Novel mutant human fibronectin FIII9-10 domain pair with increased conformational stability and biological activity

The ninth and tenth type III domains (FIII9–10) in thecentral cell binding domain of human fibronectin contain integrinreceptor binding sites, including RGD in FIII10 and a synergysite, PHSRN, in FIII9. The specific amino acids that contributeto cell binding have been identified by the use of wild-typeand mutant fragments of human fibronectin containing the FIII9–10domain pair. At high concentrations FIII9–10 mimics, toa large extent, the biological activity of the full-length fibronectinmolecule. However, FIII9 is conformationally unstable, evenin the context of the FIII9–10 pair. Here we report theconstruction of a series of hybrid mouse–human FIII9–10pairs that confer varying degrees of conformational stabilityto FIII9. The conformational stability of the human FIII9 modulewas increased 2–3-fold by substitution of Leu1408 withPro. We demonstrate that the biological activity of this mutantis enhanced. The resulting FIII9–10 mutant has good solutionproperties and will provide a template into which further mutationscan be incorporated in order to probe the structure–functionrelationship of the cell binding module of fibronectin.