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 str uctural significance of module-module interactions in the RGD-dependen t cell binding region of human fibronectin, comprising the ninth and t enth fibronectin type III. A combination of protein engineering, therm odynamics and nuclear magnetic resonance methods was employed to estab lish a relationship between intermodular protein-protein interactions and the structural properties of the module pair. A poly(glycine) pept ide 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 equil ibrium and kinetic unfolding experiments were carried out on five diff erent single and double module constructs. The results show that non-s pecific protein-protein interactions provide the bulk of the thermodyn amic stabilization and the motional constraint of the two modules. Spe cific interactions between the two modules are restricted to the wild- type module pair and decline very rapidly with the insertion of additi onal linker residues. This low level of specificity is nonetheless suf ficient to fine-tune the precise module-module orientation and to prov ide the full biological activity of the wild-type pair. This suggests that individual modules in mosaic proteins can achieve a high degree o f motional constraint and mutual stabilization without the requirement for intricate and specific interactions in the module-module interfac es. (C) 1997 Academic Press Limited.