Ubiquitin Engineering: Understanding Ubiquitin Recognition and Generating Affinity Reagents

Protein-protein interactions are necessary for virtually all biological processes. There have been tremendous efforts to document the diversity of molecular recognition, and to understand how molecular recognition occurs. The understanding of molecular interaction has also served as the foundation for designing novel protein interactions for use in therapeutics, diagnostics and basic sciences. An attractive system for studying protein-protein interactions is the ubiquitin (Ub) system. Ub is a protein modifier that is combinatorially ligated onto substrate proteins to influence substrate turnover and function. Ub uses a common surface to interact with more than 1000 proteins and plays pivotal roles in cell physiology. Despite the substantial structural information on Ub mediated interactions, there is no clear understanding of how individual Ub residues contribute to Ubâ s broad scope of interactions. To address this question, I used affinity enhanced Ub variants (Ubvs) as proxies of native Ub in saturation scanning. Using saturation scanning, I studied the interactions between Ubvs and two Ub specific proteases (USP), USP2 and USP21, and elucidated a common functional epitope that is critical for USP recognition. The functional epitope recognizes USP residues that are conserved among the human USP family, suggesting it may make functional contributions in many other USP interactions. While the functional epitopes for Ub binding to USP2 and USP21 share many identical residues, there exist distinct clusters of residues that are responsible for gaining high affinity and specificity to either USP2 or USP21. In this thesis, I also describe the usage of Ub as an alternative scaffold for generating affinity reagents. I developed a novel Ub phage library for the generation of Ubvs to targets of interest. I present validation data on Ubvs selected to bind mammalian cell surface receptor, Her3, and an intracellular protein, Grb2, showing that Ubvs can be used like conventional antibodies in cell biological experiments and can be utilized to antagonize cell signaling within a cell. My work demonstrates that protein-engineering approaches can reveal novel aspects of Ub biology and generate novel affinity reagents to probe intracellular signaling pathways.Ph.D