The binding of cadherins from apposing cell surfaces drives the development of organized multicellular structures. However, the physical basis for cadherin function, especially the differential binding specificities of the nineteen classical (type I and type II) cadherins, and the role of the unique GPI-anchored T-cadherin that functions in concert with them, remain poorly understood. The goals of this proposal are to provide a molecular, structural, and energetic view of classical cadherin function and to relate binding specificity at the molecular level to adhesive specificity at the cellular level. In the past funding period we made significant progress on these questions (a) by solving the first crystal structure of a complete type I cadherin ectodomain;(b) by solving the first crystal structures of type II cadherins;(c) by clearly establishing the role of 2-strand swapping in the EC1 cadherin ectodomain as the primary structural mechanism underlying classical cadherin binding specificity;(d) by developing in-vivo assays for type II cadherin function;(e) by showing that a single hydrogen bond can determine binding specificity between type I cadherins;and (f) by solving the first crystal structures of T-cadherin, showing that this non-classical cadherin functions by a novel mechanism. These advances provide much of the basis for the specific aims of the current proposal. We will (Aim 1) Determine binding affinities for an all-against-all matrix of classical cadherins and T-cadherin, and (Aim 2) employ our structural understanding of cadherins to design and characterize cadherin mutants with altered adhesive specificities. These studies will provide an improved understanding of the relationship between cadherin binding affinities and the adhesive behavior of cells, shedding new light on the functional implications of cadherin expression patterns observed in vertebrate development. Because cadherins are involved in the development of virtually all multicellular structures in vertebrate animals, their function and dysfunction have broad impact on human health. Mutations in cadherins are the underlying cause of many heritable defects. Loss of cadherin function, allowing tumor cells to de- adhere and become mobile in the body, is thought to be a prerequisite for metastasis of some forms of cancer. The research we propose will produce a comprehensive atomic-level understanding of cadherins that will be critical in understanding cadherin-related genetic disorders and can provide a basis for the development of small molecule drugs selective for inhibition of individual cadherins.