Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

• Published in: International journal of molecular sciences Vol. 22; no. 9; p. 4512
• Main Authors: Marcinkowski, Michał, Pilžys, Tomaš, Garbicz, Damian, Piwowarski, Jan, Mielecki, Damian, Nowaczyk, Grzegorz, Taube, Michał, Gielnik, Maciej, Kozak, Maciej, Winiewska-Szajewska, Maria, Szołajska, Ewa, Dębski, Janusz, Maciejewska, Agnieszka M, Przygońska, Kaja, Ferenc, Karolina, Grzesiuk, Elżbieta, Poznański, Jarosław
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 26-04-2021; MDPI
• Subjects: Adipogenesis; Baculovirus; BESFTO; Biomedical materials; Calcium; Calcium ions; Catalytic activity; Cell cycle; Cofactors; dimerization; ECFTO; Iron; Ketoglutaric acid; Kinases; Mimicry; Mutation; N6-methyladenosine; nanoDSF; Osteogenesis; Phosphorylation; Physiology; Proteins; Size exclusion chromatography; BESFTO; calcium; nanoDSF; ECFTO; dimerization; HDX; phosphorylation; MST; SAXS
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms22094512

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying
-methyladenosine (
-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe
/Mn
and 2-OG), Ca
that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either
(
FTO) or baculovirus (
FTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that
FTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in
FTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca
, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.