Structural and functional studies of F-box-only protein Fbxo7 and its interactions with proteasome inhibitor PI31

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

F-box only protein 7 (Fbxo7), a member of the F-box-only subfamily of FBPs, is a biologically and pathophysiologically important human protein that assumes many critical functions. The different functions of Fbxo7 depend on the formation of various multi-protein complexes. Possible interplay between different Fbxo7 functions further complicate the protein-protein interaction networks involved in Fbxo7 biology. Although significant progresses have been made to understand the functions, regulation, specificity, and protein interaction network of Fbxo7, a myriad of questions remain to be answered. The objectives of the work presented in this dissertation are to elucidate the molecular structures underlying the functions of Fbxo7 and the interaction with its protein partners, such as proteasome inhibitor PI31. The best known biological function of Fbxo7 is its role as the substrate-recognition subunit of the SCFFbxo7 (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 also assumes various SCF-independent functions through interact with its protein partners that are not the substrates of the ubiquitin proteasome system, such as PI31, Cdk6, p27, PINK1 (PTEN-induced kinase 1), and Parkin. PI31 is a known proteasome regulator which was initially characterized as a proteasome inhibitor in vitro. The binding affinity between Fbxo7 and PI31 is very strong, and The Fbxo7-PI31 interaction is mediated by heterodimerization of the FP domains of the two proteins. This work is focus on study the protein structure of the two FP domains in Fbxo7 and PI3. Chapter 1 reviewed the F-box-only protein Fbxo7 biology including the function of Fbxo7 protein in ubiquitination proteasome pathway and some SCF-independent functions which are relate to human disease. Chapter 2 discussed the function of proteasome inhibitor PI31. With the many important biological functions, Fbxo7 is clearly an extraordinary important protein, but the lack of structural knowledge has hampered efforts to achieve a better understanding of Fbxo7 biology. In this work, we have determined the crystal structure of Fbxo7 FP domain (residues 181-335) and the crystal structure of the PI31 FP domain (residues 1-161) using a longer protein construct both at 2.0Å resolution. The Fbxo7 FP domain adopts an α/β-fold similar to that of the PI31 FP domain and the secondary structure elements of the two FP domains are comparable including the C-terminal helix, indicating that the two FP domains share the same overall global fold. However, an α helix and three β strands in the Fbxo7 are longer than their counterparts in the PI31 FP domain. The two FP domains also differ substantially in the length and conformation of the longest connecting loop. More importantly, structural differences between the two FP domains lead to drastically different modes of inter-domain protein–protein interaction: the PI31 FP domain utilizes either an α interface or β interface for homodimeric interaction, whereas the Fbxo7 FP domain utilizes an αβ interface. We have note that the inter-domain interaction of the Fbxo7 FP domain is much more extensive, featuring a larger contact surface area, better shape complementarity and more hydrophobic and hydrogen-bonding interactions. The results of this structural study provide critical insights into how Fbxo7 may dimerize (or multimerize) and interact with PI31 via the FP domain. Chapter 4 and Chapter 5 discussed the structure determinations, structure features and detail of protein-protein interactions of Fbxo7 and PI31 FP domains. Chapter 2 reviewed the corresponding fundamental biochemical techniques that been used in this study. Chapter 3 discussed protein structure determination by X-ray crystallography in structural biology studies. It was believed that the FP domains of Fbxo7 and PI31 mediate homodimerization and heterodimerization of the proteins and the FP domain is not present in other human proteins. In order to study the Fbxo7-PI31 heterodimerization protein-protein interactions, we performed modeling studies. Chapter 6 discussed the model building and binding studies. Based on the result of model building studies, we propose that an interaction between the two FP domains of Fbxo7 and PI31 should be mediated by a αβ interface using the α-helical surface of the Fbxo7 FP domain and the β-sheet surface of the PI31 FP domain. According to the result of pull down assay, the PI31 FP domain may complete with Skp1 for the binding with Fbxo7. It is possible that the formation of heterodimer between the Fbxo7 and PI31 mediate by FP domains may lead to the Fbxo7 dissociation from SCFFbxo7 complex which might reveal a new regulation mechanism.