In vitro selection of fibronectin gain-of-function mutations

Directed protein evolution, which employs a combination of random mutagenesis, phage display, and in vitro selection, was used to identify second-site suppressors of the fibronectin (Fn) cell binding domain mutation Asp¹⁴⁹⁵ Ala (RGA). The mutations in the Fn 9^th (3fn9) and 10^th (3fn 10) type III repeats obtained after selection on purified integrins αIIbβ3(D¹¹⁹Y) and α5β1 are reported. The 3fn9-10(D¹⁴⁹⁵ A) phage with substitution mutations at Asp¹⁴¹⁸, which is located within the linker region between 3fn9 and 3fn10, enhanced binding to the integrins αIIbβ3 and α5β1, but not αvβ3. The substitution mutations identified at residue Asp¹⁴¹⁸ were introduced into the native recombinant 3fn9-10 sequence and found to augment binding to αIIbβ3, demonstrating that the observed gain-of-function phenotype was independent of the multivalent character of the phage. These results support the following conclusions. First, regions of Fn in addition to the RGD loop are in close proximity to αIIbβ3 and α5β1 and are capable of participating in the binding to these integrins. Secondly, the conformational relationship between the 3fn9 and 3fn10 modules may be an important factor in the binding of Fn to these two integrins. Thirdly, other altered properties of Fn-integrin interactions, such as integrin specificity, may also be selected. This is the first description of Fn mutations that augment binding to integrins. The ability to select for particular phenotypes in vitro and the subsequent characterization of these mutations should further our understanding of the molecular details involved in the association of integrins and their ligands. Additionally, these higher-affinity 3fn9-10 ligands provide a starting point for further in vitro evolution and engineering of integrin-specific modules.