Recognition by integrins of macromolecular ligands that contain Arg-Gly-Asp (RGD) sequences is a keyevent in many processes in the vasculature including hemostasis and angiogenesis, and is targeted by severaltherapeutics. This project will define the atomic basis for recognition and shape changes in integrinextracellular domains that regulate affinity for ligand and are linked by conformational transmission acrossthe plasma membrane to cytosolic signaling pathways. The structure of the integrin a,]b|33 headpiece bound tothe C-terminal y chain dodecapeptide of fibrinogen reveals binding of Lys-406 to the anb p-propeller pocketand unexpected binding of the Val-411 a-carboxyl group to the p3 MIDAS Mg21' and water-mediatedcoordination of the Asp-410 sidechain to the ADMIDAS Ca2^. A complex with the intact yC module will besought to test the hypothesis that in general, the integrin P-propeller cap subdomain recognizes foldedportions of macromolecular ligands, and an adjacent interface with the P I domain recognizes more mobilesequence motifs such as Arg-Gly-Asp and regulates affinity for ligand. Complex structures of the anbp3 andavp3 headpieces with yC module, its dodecapeptide, fibronectin FN3 modules 9 and 10, and perhaps otherRGD-containing macromolecules will reveal 1) whether avp3 and anbp3 recognize the dodecapeptide in thesame manner, 2) the generality of the two-site model for macromolecule binding described above, and 3)compare binding to these two sites of different ligands by the same integrin and different integrins to the sameligand. Structures of intact airbp3 will reveal whether the I-EGF1/I-EGF2 interface is spring loaded in anintact integrin, and reveal sites to which novel classes of antagonists that prevent integrin extension couldbind. Structures of the domains that flank the integrin knees would reveal key changes in the a and P subunitlegs that occur upon extension. At least one novel RGD-bearing ligand will be characterized structurally.The RGD site in von Willebrand factor is in the VWC repeat which is structurally unknown, and is importantin hemostasis in shear flow. In the large latent complex of TGF-P, an RGD in the latency-associated peptideis key to release of active TGF-P from storage in the extracellular matrix
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