Lynch, Andrew John2016-02-222016-02-222016https://hdl.handle.net/2144/14550This dissertation comprises identifying the structural determinants of binding selectivity as demonstrated in three systems. The first system involves the structure determination of Keap1-small molecule fragment complexes to locate binding surfaces. The second system involves the structural determination of a NEMO/IKKbeta complex to serve as a platform for future fragment binding validation studies. The third system involves the structural investigation of a bacterial phosphoglycosyltransferase found in Campylobacter concisus to find the active site. Keap1 binding of Nrf2 is a regulatory mechanism to inhibit the transcription factor activity of Nrf2 to upregulate Nucleoporin p62 (p62). Nucleoporin p62 is a regulator of tau protein aggregates in Alzheimer's disease. The determination of binding hot spots in the Keap1 active site could serve as a starting point for the development of inhibitors as a treatment method for Alzheimer’s disease. To achieve this, I have developed a crystal form of Keap1 that allows for fragment-based study of binding in the active site via small molecule fragment screening and X-ray crystallography. Analysis of collected data has resulted in the solution of four structures, one containing a peptide fragment and three containing small molecule fragments that occupy a region of binding within the Keap1 active site, demonstrating the utility of the crystal form and affording information on binding hot spots. Nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) is a transcription factor and has been linked to cancer, inflammation, and immune dysfunction. The enzyme complex IκB kinase (IKK) is a regulator of NF-κB and consists of three subunits: IKK-α, IKK-β, and NEMO. If NEMO activity is abrogated, IKK is unable to activate NF-κB, making it a promising therapeutic target. My research has found crystallization conditions and performed trials of phase determination on an N terminal IKKβ-binding construct of NEMO containing previously uncharacterized regions of this protein. Glycosylation is a commonly occurring post-translational modification that affects a number of processes including protein folding, trafficking, cell-cell interactions and host immune response. The phosphoglycosyl transferase PglC is an essential part of the Campylobacter glycosylation pathway and a possible antibacterial target. My research determined the crystallization conditions and has developed complexes and protein constructs for phase determination of this single-pass transmembrane protein and will in the future provide a platform for structure-based inhibition of this protein.en-USBiochemistryFbddKeap1NEMONrf2StructureThesis/Dissertation2016-02-13