Vinculin is a highly conserved protein that plays essential roles in directing the proper assembly of the actin cytoskeleton following the formation of focal (cell-matrix) and adherens (cell-cell) junctions. Vinculin orchestrates changes in the cytoskeleton by binding to key scaffold proteins, including talin, which binds to integrin receptors in focal adhesions, and to a-actinin and a-catenin, which bind to cadherins in adherens junctions. Previous biochemical studies have suggested that activation of vinculin is largely accomplished through severing the intramolecular association of vinculin's head (Vh) and tail (Vt) domains. This was thought to occur through the binding of acidic phospholipids to the Vt domain, which would unfurl Vt, leaving a rigid Vh domain free to associate with its binding partners. To establish the underlying principles governing vinculin activation in our Preliminary Studies we solved the crystal structures of the inactive human Vh:Vt complex, and of a talin vinculin binding site (talin VBS3) bound to Vh. The crystal structure of the Vh:Vt complex reveals that, in its resting state, vinculin is held in a closed conformation through interactions of amphipathic a-helical bundles present in Vh and Vt. More importantly, the crystal structure of talin-bound Vh has demonstrated a novel and unexpected level of control, whereby binding of talin to Vh triggers striking alterations in Vh, creating an entirely new helical bundle structure that abolishes the binding site for Vt. These findings support a model in which: 1. Talin plays a direct and active role in vinculin activation; and 2. Vh is a dynamic, flexible, domain that functions as a molecular switch which controls cytoskeletal rearrangements in adherens versus focal adhesion junctions. In support of this model, experiments in Specific Aim #1 will define the crystal structure of native, full-length vinculin and of Vh bound to vinculin binding sites present in talin. The vinculin binding domains of talin and of other vinculin-binding proteins are highly related and are predicted to bind Vh in a mutually exclusive manner. Therefore in Specific Aim #2 we will test the model that the Vh domain functions as a molecular switch that directs focal adhesions versus adherens junctions, by solving the structure of the Vh domain, and of vinculin, in complex with other vinculin binding proteins. Finally, the successful crystallization of full-length human vinculin now allows us to also determine the crystal structures of other key binding partners that interact with vinculin. Thus in Specific Aim #3 we will determine the crystal structures of vinculin bound to proteins that bind to its Proline-rich domain that links Vh and Vt. It is hoped that the proposed experiments will pinpoint the interactions that are required for proper cytoskeletal assembly in normal cells, and how these interactions may be disrupted in cancer or other disease states. [unreadable] [unreadable]