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Title: Structural and functional analysis of the proteasome inhibitor PI31 and its interaction with the F-box protein Fbxo 7
Author: Kirk, Rebecca Jane
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2005
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This thesis describes the structural and functional analysis of two related proteins, PI31 and Fbxo7, both of which act within the ubiquitin-proteasome system. This system is involved in a wide variety of cellular processes. Proteins modified with ubiquitin are often targeted for degradation by the proteasome, an ATP-dependent multi-subunit complex. Specificity for ubiquitin transfer is controlled by the E3 ubiquitin ligases. The multi-subunit SCF E3 ubiquitin ligases use their F-box domain protein to engage substrates. Analysis of possible binding partners of the novel F-box protein Fbxo7 using an affinity pull-down in Jurkat cell lysates identified PI31, a putative proteasome inhibitor. Fbxo7 was also shown to bind Skpl, Cullinl and Rbxl inferring that it forms part of a functional E3 ubiquitin ligase. Crystallisation of the Fbxo7-Skpl complex yielded poorly diffracting crystals with a maximum resolution of 8A. The PI31 N-terminal domain (NTD) shows significant sequence similarity to a globular domain of Fbxo7. The PI31-NTD structure was obtained at 2.64A by MAD phasing after introducing an additional methionine to engineer a non-centrosymmetric selenium substructure. The structure reveals a homodimer of novel a/p topology, which we define as the FP (Fbxo7 - PI31) domain since its distribution is limited to Fbxo7 and PI31 proteins in higher eukaryotes. Biophysical analysis of structure-based mutations identified the relevant residues in the homodimer interface by confirming the production of a monomeric form of PI31. Further mutations also revealed the binding surface between Fbxo7 and PI31 by using isothermal calorimetry. Fbxo7 and PI31 were shown to interact in vivo by co-immunoprecipitation and yeast two-hybrid screens using either PI31 or Fbxo7 as bait. Fbxo7 was shown to ubiquitinate FLAG-tagged PI31 in vivo. Abolishing the interaction between Fbxo7 and PI31 by mutations in PI31 prevents this ubiquitination. A model for the interaction between PI31 and Fbxo7 is presented.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available