The proposed research addresses the role of divalent cations in the recognition of fibronectin, an extracellular matrix protein, by its RGD- dependent receptor integrin alpha5beta1 at the structural level. Both macromolecules are central to the regulation of tumor cell proliferation and migration, as well as invasion and metastasis. As in other integrins, the alpha subunit of alpha5beta1 displays repeated amino-acid sequences that have a strong homology with the EF-hands present in calcium-binding proteins, except that the invariant sixth ligand, Glu, is missing in the integrins. In no absence of precise information about the folding of alpha5beta1 integrin, the tertiary structure of recombinant EF-hand fragments will be determined, in the absence and presence of divalent cations, by multidimensional NMR in solution. It has been hypothesized that the aspartyl residue of the fibronectin RGD signal plays the role of the missing ligand in one of the incomplete integrin EF-hands. This hypothesis will be tested with models which involve recombinant alpha5 EF- hands and RGD-containing fibronectin fragments, in the presence of Cd2+, in place of Ca2+, and the fibronectin-integrin contacts will be monitored by 113/Cd NMR. The conformation adopted by the RGD motif in such fibronectin fragments bound to soluble alpha5beta1 integrin will be determined in solution by two-dimensional high-resolution NMR (transferred nuclear Overhauser effect). The determination of the conformation of bound RGD sequences, as well as of the alpha5 EF-hand domain will contribute to our knowledge of the tertiary folding of the alpha5beta1 integrin and will help in the design of novel ligand analogs which could be used for further in vitro and in vivo studies on cell proliferation and migration and in the development of new anti-tumor therapies.
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