Structural and Functional Characterization of Ubiquitin Variants and Ubiquitin Binding Proteins

The ubiquitin (Ub) system is an essential post-translational modification pathway found throughout eukaryotic cells and consists of proteins responsible for adding Ub to substrates (E1, E2, and E3 enzymes), removing Ub from substrates (deubiquitinases), and reading the Ub encoded signal found on substrates. The Ub system regulates cellular functions such as DNA repair and cell-cycle progression and as such, its dysregulation can contribute to disease states. Due to its prevalent nature in regulating critical cellular functions therapeutic modulation of the Ub system has become a promising area for therapeutic development. The first step towards modulating any cellular system is target identification, which requires a detailed understanding of the system’s components and how they function. Towards this end, Dr. Sidhu and his lab have developed a set of protein-based tools termed Ub variants (UbV). These reagents are engineered variants of Ub that contain substitutions that allow them to selectively and tightly bind to specific Ub binding proteins in the cell, leading to the inhibition of and in some cases activation of the targeted protein’s function. Consequently, researchers can use UbVs to understand the role of the targeted proteins in vivo and their potential as therapeutic targets. In this thesis, I aid our understanding of the Ub system through the use of biochemical and structural biology approaches by: (i) helping further the development of UbVs tools by identifying how they bind with high specificity and affinity to their target proteins and (ii) furthering our understanding of how the deubiquitinase USP37 uses a unique insertion containing Ub interacting motifs to cleave ubiquitin chains.Ph.D

Structural and functional characterization of a ubiquitin variant engineered for tight and specific binding to an alpha-helical ubiquitin interacting motif

Ubiquitin interacting motifs (UIMs) are short α-helices found in a number of eukaryotic proteins. UIMs interact weakly but specifically with ubiquitin conjugated to other proteins, and in so doing, mediate specific cellular signals. Here we used phage display to generate ubiquitin variants (UbVs) targeting the N-terminal UIM of the yeast Vps27 protein. Selections yielded UbV.v27.1, which recognized the cognate UIM with high specificity relative to other yeast UIMs and bound with an affinity more than two orders of magnitude higher than that of ubiquitin. Structural and mutational studies of the UbV.v27.1-UIM complex revealed the molecular details for the enhanced affinity and specificity of UbV.v27.1, and underscored the importance of changes at the binding interface as well as at positions that do not contact the UIM. Our study highlights the power of the phage display approach for selecting UbVs with unprecedented affinity and high selectivity for particular α-helical UIM domains within proteomes, and it establishes a general approach for the development of inhibitors targeting interactions of this type

A molecular explanation for the recessive nature of <em>parkin</em>-linked Parkinson's disease

Mutations in the park2 gene, encoding the RING-inBetweenRING-RING E3 ubiquitin ligase parkin, cause 50% of autosomal recessive juvenile Parkinsonism cases. More than 70 known pathogenic mutations occur throughout parkin, many of which cluster in the inhibitory amino-terminal ubiquitin-like domain, and the carboxy-terminal RING2 domain that is indispensable for ubiquitin transfer. A structural rationale showing how autosomal recessive juvenile Parkinsonism mutations alter parkin function is still lacking. Here we show that the structure of parkin RING2 is distinct from canonical RING E3 ligases and lacks key elements required for E2-conjugating enzyme recruitment. Several pathogenic mutations in RING2 alter the environment of a single surface-exposed catalytic cysteine to inhibit ubiquitination. Native parkin adopts a globular inhibited conformation in solution facilitated by the association of the ubiquitin-like domain with the RING-inBetweenRING-RING C-terminus. Autosomal recessive juvenile Parkinsonism mutations disrupt this conformation. Finally, parkin autoubiquitinates only in cis, providing a molecular explanation for the recessive nature of autosomal recessive juvenile Parkinsonism.</p

Engineered SH2 Domains for Targeted Phosphoproteomics.

A comprehensive analysis of the phosphoproteome is essential for understanding molecular mechanisms of human diseases. However, current tools used to enrich phosphotyrosine (pTyr) are limited in their applicability and scope. Here, we engineered new superbinder Src-Homology 2 (SH2) domains that enrich diverse sets of pTyr-peptides. We used phage display to select a Fes-SH2 domain variant (superFes; sFe