1. The Role of Zinc in the Interaction of TAX1BP1 and Itch Studied by Fluorescence Anisotropy and Structural Modeling Ashley F. Cowan, Joel E.P. Brandis, and Barbara T. Amann Department of Biology and Chemistry, Goucher College, 1021 Dulaney Valley Rd., Baltimore, MD 21204 Abstract Zinc deficiency leads to a suppressed immune system although the mechanism is not fully understood. The Nuclear Factor kappa B (NF-kB) signaling pathway turns inflammation on and off. Many key proteins in this pathway bind to zinc. The tightest interaction occurs between of the second TAX1BP1 zinc finger (TAXF2) and the first Itch WW domain (WW1) domain when zinc is present. Through mutational studies, we have determined that four amino acids residues located in the TAXF2 are responsible for this increase affinity in the presence of zinc. Background Zinc deficiency affects 2 billion people worldwide and leads to major health problems including a suppressed immune system. The NFkB signaling pathway has many zinc binding proteins, which may explain why zinc leads to a suppressed immune system. Zinc binding proteins are necessary for turning on and off inflammation. Within the NFkB pathway, the A20 complex shuts off inflammation through negative feedback loop. TAX1BP1 and Itch are integral parts of the A20 complex. The TAX1BP1 and Itch proteins interact with one another through two PPXY zinc finger domains and four WW domains, respectively. Results Conclusion Of the TAXF1 mutants, TAXF1B2 binds the strongest to WWI and has the most similar K d value to TAXF2. TAXF1KKCPLCELMFPPNYDQSKFEEHVESHFK TAXF1b2KKCPMCEEQFPPNYDQQKFERHVQTHFD TAXF2KVCPMCSEQFPPDYDQQVFERHVQTHFD Modeling WW domain is a small three beta strand structure found in many proteins. Each TAX1BP1 zinc finger contains a PPXY domain and binds zinc with two conserved cysteines and histidines. TAX1BP1 fingers were modeled using a classical zinc finger. This causes the PPXY sequence to curve. Goal To determine what amino acids in TAXF2 allows it to bind the strongest to WWI when zinc is present. Methods Mutations were made to specific amino acids to turn TAXF1 into TAXF2: TAXF1KKCPLCELMFPPNYDQSKFEEHVESHFK TAXF1aKKCPMCELQFPPNYDQQKFEEHVETHFK TAXF1b1KVCPMCSLQFPPDYDQQVFEEHVETHFK TAXF1b2KKCPMCEEQFPPNYDQQKFERHVQTHFD TAXF2KVCPMCSEQFPPDYDQQVFERHVQTHFD Experimental Procedure CC1CC2CC3 PPXY ZF1 PPXY ZF2 C2 HECTWW1WW2WW3WW4 TAX1BP1 ITCH RNF11 A20 A20 Complex includes the proteins RNF11, Itch, TAX1BP1, and A20 WW domainClassic TFIIA zinc finger and PPXY region unattached Model of TAXF2 zinc finger with PPXY region in red Synthetic peptides purified by HPLC. Binding activity of cobalt to protein quantified by uv/vis spectroscopy. Cobalt then substituted by zinc. K d values of zinc fingers bond to WWI determined from fluorescence anisotropy. Figure 1. Anisotropy compared to the concentrations of TAXF2 or mutants of TAXF2 when added to WWI. Graph produced through the use of KaleidaGraph. The amino acids in red are those determined to be important to the interaction of TAXF2 and WWI. Future experiments could lead to us discovering the specific amino acid(s) responsible for the increased bonding. Table 1. K d values in  M of TAX1BP1 domains TAXF2 and TAXF1 with mutations bond to Itch WW1. All TAX1BP1 domains contain zinc. K d values are averaged from 2-3 different trials. + Zinc