The generation of a lethal tumor mass requires that the tumor cells recruit and sustain their own blood supply. In the absence of blood supply, tumors can persist as dormant microscopic lesions but they do not expand beyond the size of 1-2 mm. Hence, inhibition of new blood vessel growth (angiogenesis) offers great promise to eradicate tumors or to convert cancer to a chronic manageable disease. In addition, inhibition of angiogenesis offers great promise to treat a range of other diseases that depend on angiogenesis, including age-related macular degeneration, diabetic retinopathy, rheumatoid arthritis and atherosclerosis. The focus of this application is anastellin, a fragment of the extracellular matrix protein fibronectin that inhibits angiogenesis, tumor growth and metastasis in mouse models. Anastellin requires endogenous fibronectin for its in vivo anti- angiogenic activity, and it binds to fibronectin in vitro and converts the soluble protein to insoluble fibril. Our long term goal is to elucidate how the interaction of anastellin with fibronectin leads to formation of fibrillar aggregates, and in general how the structure of fibronectin determines its function. The goal of this application is to identify the molecular basis of the interaction betwee anastellin and fibronectin. We propose to investigate the structure and dynamics of the complexes between anastellin and its target fibronectin type III (FN3) domains by disulfide crosslinking, nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. In addition, we propose to investigate the conformation of the anastellin binding region in fibronectin using paramagnetic relaxation enhancement (PRE) and single molecule Frster resonance energy transfer (FRET). Identification of structural features that are responsible for the activity of anastellin will provide detailed understanding of how this inhibitor of angiogenesi acts at the molecular level and may enable development of new anticancer drugs. The proposed structural studies will also provide insight into the molecular interactions and rearrangements that are involved in conversion of soluble fibronectin to the fibrillar matrix form.