Researcher(s)
- Rebecca Payne, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Jeffrey Mugridge, Chemistry and Biochemistry, University of Delaware
Abstract
Fat mass and obesity-associated protein (FTO) belongs to the iron(II)/α-ketoglutarate-dependent family of dioxygenases and plays an important role in regulating gene expression. FTO is an RNA demethylase that removes N6,2′-O-dimethyladenosine (m6Am) and N6-methyladenosine (m6A) modifications from RNA. These modifications are widespread in the transcriptome and influence RNA stability, translation, and processing. Dysregulation of FTO activity has been linked to several human diseases, including obesity, cancer, and metabolic disorders. Although the apo structure of FTO has been determined in previous studies, FTO preferentially crystallizes in a single crystal lattice that creates significant steric clashes with nucleic acid substrates. As a result, obtaining a structure of FTO bound to nucleic acids has been extremely challenging.
Multiple lysozyme-FTO fusion constructs (32-505, C-terminal, and Δ Loop) were designed to promote new crystal packing interactions. In tandem with the lysozyme fusion constructs, Maltose Binding Protein (MBP) was also used to create fusion proteins with the full-length FTO and the N-terminal domain. These were overexpressed in E. coli BL21 cells and purified by affinity, ion-exchange, and size-exclusion chromatographies, where SDS-PAGE was utilized to assess the purity of the constructs. The lysozyme fusion constructs were screened against commercially available crystallography conditions. The promising conditions were then optimized to promote better crystal growth and quality, and crystals were sent to the Brookhaven National Laboratory NSLS-II synchrotron beamline for data collection and structure determination. In the future, more fusion constructs will be produced, and the fusion FTO structures will be solved using molecular replacement and refined in Phenix to generate an atomic-level model of the complex.