Module-module interactions in the cell binding region of fibronectin: Stability, flexibility and specificity

The structure of mosaic proteins depends on the nature and strength of interactions between individual modules. Here we investigated the structural significance of module-module interactions in the RGD-dependent cell binding region of human fibronectin, comprising the ninth and tenth fibronectin type III. A combination of protein engineering, thermodynamics and nuclear magnetic resonance methods was employed to establish a relationship between intermodular protein-protein interactions and the structural properties of the module pair. A poly(glycine) peptide link connecting the C terminus of the ninth and the N terminus of the tenth module was introduced to probe the range of the interaction. NMR studies (Chemical shifts and N-15 relaxation) together with equilibrium and kinetic unfolding experiments were carried out on five different single and double module constructs. The results show that non-specific protein-protein interactions provide the bulk of the thermodynamic stabilization and the motional constraint of the two modules. Specific interactions between the two modules are restricted to the wild-type module pair and decline very rapidly with the insertion of additional linker residues. This low level of specificity is nonetheless sufficient to fine-tune the precise module-module orientation and to provide the full biological activity of the wild-type pair. This suggests that individual modules in mosaic proteins can achieve a high degree of motional constraint and mutual stabilization without the requirement for intricate and specific interactions in the module-module interfaces. (C) 1997 Academic Press Limited.