Open Access Dissertation
Date of Award
Biochemistry and Molecular Biology
X-ray crystallography is the gold standard method for imagining macromolecules to atomic resolution. Three dimensional data is central to understanding the molecular mechanism how DNA, RNA and proteins function in biological events. Structural insights into these events provide a molecular window to visualize how biological molecules influence human health. Visualizing the architecture of these molecules set the stage for rational and selective drug design. The following dissertation utilizes biochemical and biophysical tools, including X-ray crystallography, to shed light on poorly understood mechanisms related to SMYD2 activity and regulation, USP10 architecture and function, and PDZ-RhoGEF dimerization. SMYD2 is one member of the SET and MYND domain-containing protein (SMYD) family known to play key roles in cardiac function and development, innate immunity and tumorigenesis. While the molecular pathways involved in these events have been fairly described, the molecular mechanism of substrate recognition and bilobal changes have not. In this dissertation, I review the structure and function of SMYD protein family. In addition, I demonstrate SMYD2 and SMYD3 can exist in open and closed conformations based on X-ray crystallography, small angle X-ray scattering, and molecular dynamic simulations data. Lastly, I revealed a novel binding site in SMYD2 that appears to be the first recognition site for SMYD methylation clients. USP10 is one member of the ubiquitin-specific protease family important for DNA repair and apoptosis by recycling cytosolic p53. However, in the mutant p53 environment, USP10 serves as an oncogene; thereby promoting mutant p53-dependent cancer cell growth. Additional studies found related USP10 oncogene roles in other cancers. Unfortunately the biochemistry and structure of USP10 hasn’t been thoroughly explored. My dissertation aims to understand the biochemistry and architecture of the catalytic domain of USP10 along with reported USP10 inhibitors which would be valuable for future studies to probe USP10 function and inhibition. PDZ-RhoGEF is one member of the Rho guanine exchange factors (RhoGEF) family important for modulating Rho activity and actin-based cytoskeleton remodeling. PDZ-RhoGEF possesses a PDZ domain known for complexing with the cytoplasmic tail of Plexin B serving as modulator for downstream signaling factors. In our study, we found PDZ-RhoGEF complexes with the Interleukin-8 chemokine receptor, CXCR2. This novel interaction hasn’t been reported before, and in my dissertation, I solved the crystal structure of PDZ-RhoGEF in complex with the PDZ motif of CXCR2. Unexpectedly, we identified a disulfide bond linking two PDZ-RhoGEF molecules. This disulfide bond was previously reported to be important for promoting PDZ-ligand binding between PDZ-RhoGEF and Plexin B2 peptides. Here, I describe the architecture of the disulfide-linked PDZ domain of PDZ-RhoGEF in complex with two CXCR2 PDZ-motifs.
Spellmon, Nicholas, "Alternative Strategies To Inhibit Lysine Methyltransferases And Deubiquitinases In Human Cancers" (2018). Wayne State University Dissertations. 2073.