This 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.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/14550 |
| Date | 13 February 2016 |
| Creators | Lynch, Andrew John |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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