Abstract Zinc deficiency leads to a suppressed immune system although the mechanism is not fully understood.

1. PPXY ZF1 PPXY ZF2 CC1 CC2 CC3 + Zinc WW1 WW2 WW3 WW4 HECT C2 • 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: • TAXF1 KKCPLCELMFPPNYDQSKFEEHVESHFK • TAXF1a KKCPMCELQFPPNYDQQKFEEHVETHFK • TAXF1b1 KVCPMCSLQFPPDYDQQVFEEHVETHFK • TAXF1b2 KKCPMCEEQFPPNYDQQKFERHVQTHFD • TAXF2 KVCPMCSEQFPPDYDQQVFERHVQTHFD • Experimental Procedure • 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. Results The Role of Zinc in the Interaction of TAX1BP1 and Itch Studied by Fluorescence Anisotropy and Structural ModelingAshley F. Cowan, Joel E.P. Brandis, and Barbara T. AmannDepartment of Biology and Chemistry, Goucher College, 1021 Dulaney Valley Rd., Baltimore, MD 21204 WW domain Classic TFIIA zinc finger and PPXY region unattached Model of TAXF2 zinc finger with PPXY region in red • 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. Figure 1. Anisotropy compared to the concentrations of TAXF2 or mutants of TAXF2 when added to WWI. Graph produced through the use of KaleidaGraph. Table 1. Kd values in mM of TAX1BP1 domains TAXF2 and TAXF1 with mutations bond to Itch WW1. All TAX1BP1 domains contain zinc. Kd values are averaged from 2-3 different trials. • Conclusion • Of the TAXF1 mutants, TAXF1B2 binds the strongest to WWI and has the most similar Kdvalue to TAXF2. • TAXF1 KKCPLCELMFPPNYDQSKFEEHVESHFK • TAXF1b2 KKCPMCEEQFPPNYDQQKFERHVQTHFD • TAXF2KVCPMCSEQFPPDYDQQVFERHVQTHFD ITCH RNF11 TAX1BP1 A20 A20 Complex includes the proteins RNF11, Itch, TAX1BP1, and A20 • 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. Binding activity of cobalt to protein quantified by uv/vis spectroscopy. Cobalt then substituted by zinc. Kd values of zinc fingers bond to WWI determined from fluorescence anisotropy. Synthetic peptides purified by HPLC.