The kinase-inducible domain interacting (KIX) domain of the coactivator CBP/p300 is involved in the regulation of hundreds of genes, some of which have applications in memory formation, hematopoiesis and the inflammatory response. Despite its wide array of applications, only one crystal structure of the KIX domain has been reported using a tethered small molecule, and none in its unliganded state. The absence of such structures are in part due to the intermediate stability of the protein. Previous research findings show that incorporating fluorinated amino acids stabilizes the protein, but the reason for this remains unknown. The focus of this project is to tether a small molecule to a mutated cysteine on KIX, and incorporate fluorinated tyrosine residues into the protein. Here, we demonstrate that a 2-fluorotyrosine and 3-fluorotyrosine-containing-KIX mutant (L664C) can be expressed, is well folded, and can be tethered with a synthetic small molecule 1-10. Fluorine NMR was utilized to verify the incorporation of the unnatural amino acids. After tethering molecule 1-10 to 3FY KIX L664C, the resonance corresponding to Y631 was perturbed. This result, as well as mass spectrometry, has verified that the molecule was successfully tethered. Stability and crystallography studies are underway. Additionally, in future work, we plan to co-crystallize the molecule tethered to KIX with other reported small molecules to potentially identify a new ligand binding site. Our study will ultimately allow for a greater understanding of the structure and stability of the KIX domain.

Structural determination of <sup>19</sup>F labeled KIX via disulfide tethering

2019 Fall National Meeting

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Structural determination of 19F labeled KIX via disulfide tethering

Structural determination of ¹⁹F labeled KIX via disulfide tethering