The complicated task of interpreting the many ubiquitin signals is mediated by specific ubiquitin binding domains. The investigations discussed in this thesis focus on two very different ubiquitin binding domains. Chapters 3 to 5 detail the structural characterisation of the ubiquitin binding protein ZNF216. The structure of the ubiquitin binding Znf_A20 domain has been determined using multidimensional NMR techniques. A thermodynamic and structural characterisation of the interaction between the Znf_A20 and ubiquitin has been performed utilising chemical shift mapping, PRE based approaches, ESI-MS and ITC. The Znf_A20 domain forms a high affinity complex with Ub utilising a non-canonical binding site on ubiquitin centred at Asp58. The investigation was extended to the function of the Znf_A20 domain in the context of the full length protein. ZNF216, like many other ubiquitin receptors, has a ‘hook and line’ domain architecture with two independent domain separated by a long disordered linker. Chapters 6, 7 and 8 focus on the UBA domain of p62. The p62-UBA domain has been identified as a ‘hot spot’ for mutations linked to Paget’s disease of bone, a bone disorder which affects >3% of the over 55s. The dimerisation of the p62-UBA domain has been shown here to be a novel regulatory mechanism for the ubiquitin binding properties of p62. Modulation of both dimerisation and ubiquitin recognition are potential mechanisms by which mutations may disrupt p62 function and this prospect has been investigated here. The final chapter of this thesis examines the possibility of cooperation between different ubiquitin binding domains by simultaneous interaction with Ub to form ternary complexes. Using the Znf_A20 and the p62-UBA as an example the formation of a ternary complex has been demonstrated. By examining the available ubiquitin complexes it has been suggested that the formation of Ub mediated ternary complexes is limited to only a few UBD pairings. The cormation of Ub mediated ternary complexes may have interesting implications for the formation of larger multi-protein complexes utilising ubiquitin as an interaction hub on the recognition of poly-Ub with chain linkage specificity; and for Ub mediated signalling in general.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:547272 |
| Date | January 2011 |
| Creators | Garner, Thomas Peter |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/12100/ |
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