The Role of Zinc in the Interaction of TAX1BP1 and Itch Studied by Fluorescence Anisotropy and Structural Modeling


Students: Ashley Cowen, Joel Brandis (Dept. of Chemistry)

Faculty Adviser: Barbara T. Amann (Dept. of Chemistry)

Abstract

Inflammation is the first line of defense of the innate immune system when the body is under attack by a foreign body.  The efficacy of inflammation has been seen to work in tandem with an appropriate intake of zinc. In the Nuclear Factor Kappa B (NFkB) signaling pathway, a number of zinc binding proteins are necessary for turning on and off inflammation and are critical for propagating the inflammation response in the innate immune system.  Within the NFkB signaling pathway, the negative regulation is controlled by the A20 complex, which consists of four proteins including Tax1 Binding Protein 1 (TAX1BP1) and the E3 ligase protein, Itch. The Itch protein contains four WW domains, which are known to interact with a small amino acid sequence PPXY where X can be any amino acid.  Intriguingly, TAX1BP1 protein contains two PPXY domains located inside of two zinc fingers. We have found that the tightest binding between these domains occurs between the TAX1BP1 second finger domain (TAXF2) bond to zinc and the first WW domain (WW1) with an overall dissociation constant, Kd, of 51 mM.   In contrast, the tightest binding of the first finger of TAX1BP1 (TAXF1) also occurs with WW1 but in the zinc free form and with a four fold weaker affinity. Through a series of peptide mutants, the amino acids responsible for this increase in binding affinity of the WW1 domain to the zinc-bound TAXF2 domain have been narrowed to four positions.  A molecular model was built of the zinc bound TAXF2 and WW1 to further clarify their interactions.

Click here for the research poster presented at the Fifth Annual Landmark Conference Summer Research Symposium