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Title page for ETD etd-07052011-193608

Type of Document Dissertation
Author Nordquist, Kyle Andrew
Author's Email Address ka.nordquist@gmail.com
URN etd-07052011-193608
Title Structural and functional analysis of the U-box domain of the E4B ubiquitin ligase
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Walter Chazin Committee Chair
BethAnn McLaughlin Committee Member
Daniel Liebler Committee Member
Tina Iverson Committee Member
Zu-Wen Sun Committee Member
  • proteasome
  • degradation
  • Ufd2
  • E4B
  • U-box
  • ligase
  • E3
  • ubiquitin
  • protein structure
  • NMR
Date of Defense 2011-03-22
Availability unrestricted
Ubiquitination is a post-translational modification, that functions in a variety of cellular signaling pathways. Unlike modification with a small functional group by a single enzyme, modification by the small protein ubiquitin requires a three-step enzymatic cascade involving E1 activating, E2 conjugating, and E3 ligating enzymes. While much information is known about this process, a comprehensive understanding of the molecular mechanisms of ubiquitination is lacking. This thesis investigated the structure and the function of the U-box E3 ligase, E4B, focusing on the interaction with the E2 enzyme and the activation of the E2~Ub conjugate by the E3 ligase.

In order to study E4B interaction with the E2 conjugating enzyme UbcH5c, I determined the three-dimensional solution structure of the U-box domain of E4B (E4BU) by NMR spectroscopy. Then, NMR chemical shift perturbation analysis was used to map the interaction with UbcH5c. This data, along with the new E4BU structure and the known structure of UbcH5c, was used to generate a computational model of the E4BU-UbcH5c complex. The oligomerization state of E4BU was also investigated; unlike other U-box E3 ligases, it was found that E4BU is monomeric. To determine if E4BU was functional as a monomeric E3 ligase, an in vitro autoubiquitination assay with UbcH5c was developed. These experiments showed that E4BU does function as a monomer.

The favorable characteristics of E4BU (small size, soluble, excellent NMR spectrum) make it an excellent model system to study the mechanism of E3 activation of the E2~Ub conjugate. Consequently, NMR chemical shift perturbation analysis was applied to the E4BU-UbcH5c~Ub complex. These results suggested the E2~Ub conjugate is activated by the E3 through an allosteric network. This model was tested by mutations made within this network, which significantly inhibited ubiquitination function. Initial studies of the dynamics of the UbcH5c~Ub conjugate in the absence and presence of E4BU were also undertaken, setting the stage for more detailed understanding of the role of dynamics in E3 activation of the E2~Ub conjugate.

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