PROJECT SUMMARY The human skin barrier does not always function as intended; in fact, defects in the outer layer of human skin account for a significant number of health problems for people living in the United States. As one example, it is estimated that up to 90 million Americans suffer from some form of atopic dermatitis. Atopic dermatitis and other forms of severely dry skin, such as ichthyosis vulgaris, are associated with defects or mutations in profilaggrin and its processed fragment, filaggrin. Mutations in keratin 1 and keratin 10 proteins also account for a variety of inherited skin disorders manifested by red, dry, scaly skin. Together, profilaggrin and keratins 1 and 10 are critical proteins involved in the stratum corneum of human skin, and elucidating the biochemistry behind their function is important for improving our understanding of how the human skin barrier works. The hope is that advancing our knowledge of how protein structure dictates function in the stratum corneum will generate new methods for treatment of human skin diseases. This proposal aims to address a deficiency in our understanding of the atomic resolution structure of key epidermal proteins, namely profilaggrin and keratins 1 and 10. In particular, it is still unclear (1) what the precise mechanisms are for profilaggrin binding to target proteins; (2) is there correlation between structure and function of the profilaggrin B domain; (3) does profilaggrin interaction with target proteins direct specific events in terminal epidermal differentiation; (4) what are the molecular mechanisms behind keratin intermediate filament aggregation; and (5) can enhanced structural knowledge of key skin barrier proteins pave the way for newly designed topical therapeutics. Given the direct association of profilaggrin and keratins 1 and 10 with multiple human diseases, we believe focusing our studies on these medically important proteins will help us address these outstanding questions. In this project, we examine the biochemical and structural properties of key proteins involved in a functional human skin barrier. Our first aim uses x-ray crystallography and biochemical analysis techniques to determine the structural basis of binding between profilaggrin and one of its targets, annexin II. The second aim examines the x-ray crystal structure of profilaggrin in the absence of bound molecules and performs mutational analysis on key inter-EF-hand linker residues in order to understand the functional role of this protein region. The third aim examines the structural basis for keratin intermediate filament interaction with different parts of the profilaggrin molecule using x-ray crystallography and biochemical techniques. Accomplishing these aims will provide novel insight into the principle biochemistry and atomic resolution structure of key epidermal proteins involved in maintaining the integrity of the human skin barrier.