Investigating role of conformational dynamics in substrate selectivity and catalysis in human RNA demethylase FTO
Date
2022-05
Authors
Khatiwada, Balabhadra
Major Professor
Advisor
Venditti, Vincenzo
Anand, Robbyn K
Nelson, Scott W
Potoyan, Davit
Underbakke, Eric S
Committee Member
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Abstract
Various recent studies have shown that conformational dynamics often regulates different aspects of enzyme function including substrate recognition, catalysis, product release and allosteric regulation. Despite of this knowledge, discerning the mechanism of conformational dynamics and its relationship to enzyme function is challenging and demands rigorous biophysical characterization. Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful tools available to study protein dynamics. Solution NMR in particular is the preferred tool to quantify dynamics due to its unique ability of providing thermodynamic and structural information on dynamics at multiple time regimes with atomic resolution.
Fat mass and obesity associated (FTO) protein is a eukaryotic, multi-domain enzyme whose overexpression has been linked to the development of obesity, type 2 diabetes, and various types of cancers in humans. Pharmacological inhibition of FTO protein is emerging as a promising strategy to develop a therapeutic treatment for obesity and cancers. Indeed, FTO has been targeted for structure-based drug design and subsequently, several competitive inhibitors of FTO have been generated. These inhibitors often lack selectivity or potency due structural similarity of FTO’s catalytic site to various other eukaryotic enzymes. Therefore, an atomic- resolution structural and functional understanding of FTO is required as it can lead to novel, alternative strategies for identifying binding sites allosterically coupled to the active site required to design highly specific, ponent FTO inhibitors.
FTO is classified as a member of non-heme Fe (II) and alpha-ketoglutarate dependent
Alkb family of dioxygenases and catalyzes the oxidative demethylation of various N-methylated
nucleobases, such as N6-methyladenosine (m6A), N1-methyladenosine (m1A), 3-methylthymidine
(m3T), 3-methyluridine (m3U) and 5’caped N6 , 2’-O-di-methyladenosine (m6Am) in eukaryotic
ssRNA and ssDNA. Previous studies have shown that Alkb family of enzymes are inherently dynamic, and the conformational dynamics plays an important role in the catalysis and product release.
My work presented in this dissertation involves the use of a combination of biophysical, biochemical, and computational methods to understand the role of conformational dynamics in the various aspects of FTO function. Herein, we utilize NMR experiments and Molecular Dynamics (MD) simulations to identify the dynamic regions of protein that play an important role in the catalysis. Additionally, we show that the dynamic interdomain interaction is essential for FTO’s function. Further, we combine NMR, enzyme kinetics and ligand-binding assays to study the substrate binding and differential activity of FTO towards its various substrates and show that the changes in conformational dynamics between the apo and holo forms govern the substrate selectivity. Finally, we identify the druggable transient pockets on the dynamic interdomain interface of FTO that can be targeted for the screening highly specific, allosteric inhibitors. This work provides a structural basis for inhibitor design in the future targeting allosteric pockets to modulate the activity for the therapeutic treatment of obesity and cancer.
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dissertation