Date of Award
Doctor of Philosophy (PhD)
Microbiology, Immunology, and Biochemistry
Brenda A. Schulman, Ph.D.
John V. Cox, Ph.D. Eric J. Enemark, Ph.D. Roderick T. Hori, Ph.D. Stephen White, Ph.D.
Tetratricopeptide repeats, APC3, Hybrid TPR, E3 ligase, 26S proteosome
Tetratricopeptide (TPR) repeats are a 34-residue helix-turn-helix motif that when repeated pack into a superhelical structure. TPR domains are frequently found mediating protein-protein interactions, often through a central groove. One protein complex bearing numerous TPR repeats is the Anaphase Promoting Complex (APC). The anaphase-promoting complex (APC) is a multi-subunit complex, which orchestrates mitotic cell cycles. APC is an E3 ligase in the ubiquitin cascade, and directs the 26S proteosome degradation of cell-cycle regulators. Throughout mitotic progression, proteins that are key regulators of the cell cycle are assembled with polyubiquitin chains by APC.
One domain of the human APC is comprised of four related TPR proteins, APC8, APC6, APC3, and APC7, with each found in pairs. Crystal structures of some of these indicate that each has an N-terminal dimerization domain and a C-terminal domain that APC3 extends away from the dimer interface. The TPR C-terminal domains are thought to play major roles in mediating protein interactions within the APC.
The subunit APC3 plays major roles in regulating APC function. Within an APC3 dimer, each C-terminal domain recruits the Ile-Arg motifs of substrate coreceptors Cdh1 (or Cdc20) and APC10. Cdh1 and APC10 together recruit substrates for ubiquitination. Therefore, it is important to understand the structure of APC3, and how APC3 mediates interactions. To address this problem, I used a novel “hybrid TPR” technology, in which some TPRs from a distant relative of APC3 are fused upstream of the C-terminal domain from human APC3. This approach enabled determination of a 3Å resolution structure encompassing the sequence of the APC3 C-terminal domain. Interestingly, only a fraction of the structure resembles canonical TPR repeats. Interpretation of the crystal structure based on published structures of complexes between TPR proteins and their partners, and on published electron microscopy structures of APC-Cdh1-APC10, reveal that the region containing the Cdh1/APC10 binding site adopts 3 canonical TPR repeats. The remainder of the portion of the structure corresponding to human APC3 is folded into an alternative conformation, in which a helix from the atypical portion of APC3 buries the Cdh1/APC10-interacting groove within the crystal. Accordingly, unlike wildtype APC3, the hybrid TPR APC3 fails to bind Cdh1 and APC10. Nonetheless, the crystal structure of “hybrid TPR APC3 C-terminal domain” allows the prediction of potentially important residues for binding to Cdh1 and APC10. Taken together, the data reveal strengths and weaknesses of hybrid TPR technology for obtaining structural insights into TPR subunits of multiprotein assemblies such as APC.
Yu, Shanshan , "Strengths and Weaknesses of Hybrid TPR Technology for Obtaining Structural and Mechanistic Insights into TPR Proteins" (2014). Theses and Dissertations (ETD). Paper 400. http://dx.doi.org/10.21007/etd.cghs.2014.0368.