The cell surface Notch receptor and its ligand, presented on adjacent cells, bind through their extracellular regions and lead to the release of Notch intracellular domain (NICD). NICD then translocates to the nucleus to form the Notch transcription activating complex. ANK domain of NICD plays an essential part in the formation of this complex and the role of ANK domain was analysed both structurally and functionally using site directed mutagenesis. A single residue E2072 on the sixth ANK repeat was found crucial to maintain the stability of the Notch transcription activating complex. It can therefore be considered as a putative “hot-spot” for the purpose of drug discovery. Recently the interaction of Neutralized (an E3 ubiquitin ligase) with Tom (a Bearded protein) was identified. This study was undertaken to understand and structurally characterise the Tom-Neuralized interaction. Expression constructs were designed using the information from sequence analysis of Tom and Neuralized proteins and expressed using bacterial expression system. The NHR1 domain, expressed as an N-terminal His-tag protein, was used to determine the crystal structure of NHR1 domain. The structure was found to be structurally related to SPRY proteins consisting of jelly roll topology. Potential binding sites were predicted that localised on a cluster referred as ‘hydrophobic patch’ in this study. The expression of Tom remained insoluble under most experimental conditions and therefore a 12 amino acid peptide was synthesized. Biophysical analysis showed the binding affinity between NHR1 and Tom was weak but still could form the complex. Comparison of NHR and SPRY domain proteins suggested that the binding interface of the two could be common indicating that the interaction of Delta could require the same binding interface of NHR1 domain as Tom. Lastly, interaction of NHR1 with PI4P was analysed and verified using interfacial studies, which showed that NHR1 protein localised below the lipidic layer.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:599795 |
| Date | January 2009 |
| Creators | Gupta, D. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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