Solution structure ensemble of human obesity-associated protein FTO reveals druggable surface pockets at the interface between the N- and C-terminal domain
2022; Elsevier BV; Volume: 298; Issue: 5 Linguagem: Inglês
10.1016/j.jbc.2022.101907
ISSN1083-351X
AutoresBalabhadra Khatiwada, Trang Nguyen, Jeffrey A. Purslow, Vincenzo Venditti,
Tópico(s)Cancer-related gene regulation
ResumoThe fat mass and obesity-associated FTO protein catalyzes demethylation of the N6-methyladenosine, an epigenetic mark that controls several metabolic pathways by modulating the transcription, translation, and cellular localization of RNA molecules. Since the discovery that its overexpression links to the development of obesity and cancer, FTO was the target of screening campaigns and structure-based drug design efforts. Although several FTO inhibitors were generated, these often lack potency or selectivity. Herein, we investigate the structure and dynamics of human FTO in solution. We show that the structure of the catalytic N-terminal domain is unstable in the absence of the C-terminal domain, which explains why the isolated N-terminal domain is incompetent for catalysis and suggests that the domain interaction represents a target for the development of specific inhibitors. Then, by using NMR relaxation measurements, we show that the interface between the FTO structural domains, the active site, and several peripheral loops undergo conformational dynamics on both the picosecond–nanosecond and microsecond–millisecond timescales. Consistent with this, we found that the backbone amide residual dipolar couplings measured for FTO in phage pf1 are inconsistent with the static crystal structure of the enzyme. Finally, we generated a conformational ensemble for apo FTO that satisfies the solution NMR data by combining the experimental residual dipolar couplings with accelerated molecular dynamics simulations. Altogether, the structural ensemble reported in this work provides an atomic-resolution model of apo FTO and reveals transient surface pockets at the domain interface that represent potential targets for the design of allosteric inhibitors. The fat mass and obesity-associated FTO protein catalyzes demethylation of the N6-methyladenosine, an epigenetic mark that controls several metabolic pathways by modulating the transcription, translation, and cellular localization of RNA molecules. Since the discovery that its overexpression links to the development of obesity and cancer, FTO was the target of screening campaigns and structure-based drug design efforts. Although several FTO inhibitors were generated, these often lack potency or selectivity. Herein, we investigate the structure and dynamics of human FTO in solution. We show that the structure of the catalytic N-terminal domain is unstable in the absence of the C-terminal domain, which explains why the isolated N-terminal domain is incompetent for catalysis and suggests that the domain interaction represents a target for the development of specific inhibitors. Then, by using NMR relaxation measurements, we show that the interface between the FTO structural domains, the active site, and several peripheral loops undergo conformational dynamics on both the picosecond–nanosecond and microsecond–millisecond timescales. Consistent with this, we found that the backbone amide residual dipolar couplings measured for FTO in phage pf1 are inconsistent with the static crystal structure of the enzyme. Finally, we generated a conformational ensemble for apo FTO that satisfies the solution NMR data by combining the experimental residual dipolar couplings with accelerated molecular dynamics simulations. Altogether, the structural ensemble reported in this work provides an atomic-resolution model of apo FTO and reveals transient surface pockets at the domain interface that represent potential targets for the design of allosteric inhibitors. FTO is a member of the Alkb family of nonheme Fe(II)- and α-ketoglutarate (αKG)-dependent dioxygenases and catalyzes oxidative demethylation of single-stranded RNAs via two coupled reactions, referred to as the primary and secondary reaction, respectively (1Jia G. Fu Y. Zhao X. Dai Q. Zheng G. Yang Y. Yi C. Lindahl T. Pan T. Yang Y.-G. He C. N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.Nat. Chem. Biol. 2011; 7: 885-887Crossref PubMed Scopus (1966) Google Scholar, 2Martinez S. Hausinger R.P. Catalytic mechanisms of Fe(II)- and 2-Oxoglutarate-dependent oxygenases.J. Biol. Chem. 2015; 290: 20702-20711Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 3Zheng G. Fu Y. He C. Nucleic acid oxidation in DNA damage repair and epigenetics.Chem. Rev. 2014; 114: 4602-4620Crossref PubMed Scopus (61) Google Scholar). In the secondary reaction, the αKG (secondary substrate) is reduced to succinic acid and carbon dioxide, while the metal center is oxidized to form an Fe(IV)=O species. In the primary reaction, the oxyferryl species oxidizes the methylated base (primary substrate) to reestablish the canonical nucleic acid. Although FTO was reported to be active against several methylated nucleobases, including the 3-methyluracil (4Jia G. Yang C.-G. Yang S. Jian X. Yi C. Zhou Z. He C. Oxidative demethylation of 3-methylthymine and 3-methyluracil in single-stranded DNA and RNA by mouse and human FTO.FEBS Lett. 2008; 582: 3313-3319Crossref PubMed Scopus (322) Google Scholar), 3-methylthymidine (4Jia G. Yang C.-G. Yang S. Jian X. Yi C. Zhou Z. He C. Oxidative demethylation of 3-methylthymine and 3-methyluracil in single-stranded DNA and RNA by mouse and human FTO.FEBS Lett. 2008; 582: 3313-3319Crossref PubMed Scopus (322) Google Scholar), 1-methyladenosine (5Wei J. Liu F. Lu Z. Fei Q. Ai Y. He P.C. Shi H. 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Structural insights into FTO's catalytic mechanism for the demethylation of multiple RNA substrates.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 201820574Google Scholar), and the N6-methyladenosine (m6A), recent evidences suggest the m6A and cap m6Am in mRNA, m6A and m6Am in small nuclear RNA, and 1-methyladenosine in tRNA as the physiological substrates of the enzyme (5Wei J. Liu F. Lu Z. Fei Q. Ai Y. He P.C. Shi H. Cui X. Su R. Klungland A. Jia G. Chen J. He C. Differential m6A, m6Am, and m1A demethylation mediated by FTO in the cell nucleus and cytoplasm.Mol. Cell. 2018; 71: 973-985.e5Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 6Mauer J. Luo X. Blanjoie A. Jiao X. Grozhik A.V. Patil D.P. Linder B. Pickering B.F. Vasseur J.J. Chen Q. Gross S.S. Elemento O. Debart F. Kiledjian M. Jaffrey S.R. Reversible methylation of m(6)Am in the 5' cap controls mRNA stability.Nature. 2017; 541: 371-375Crossref PubMed Scopus (539) Google Scholar, 7Zhang X. Wei L.-H. Wang Y. 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The atomic-resolution structure of FTO has been deeply investigated by X-ray crystallography, and several crystal structures of FTO in complex with a variety of substrate analogs and inhibitors are available in the Protein Data Bank (PDB) (7Zhang X. Wei L.-H. Wang Y. Xiao Y. Liu J. Zhang W. Yan N. Amu G. Tang X. Zhang L. Jia G. Structural insights into FTO's catalytic mechanism for the demethylation of multiple RNA substrates.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 201820574Google Scholar, 14Han Z. Niu T. Chang J. Lei X. Zhao M. Wang Q. Cheng W. Wang J. Feng Y. Chai J. Crystal structure of the FTO protein reveals basis for its substrate specificity.Nature. 2010; 464: 1205-1209Crossref PubMed Scopus (275) Google Scholar, 15Aik W. Demetriades M. Hamdan M.K. Bagg E.A. Yeoh K.K. Lejeune C. Zhang Z. McDonough M.A. Schofield C.J. Structural basis for inhibition of the fat mass and obesity associated protein (FTO).J. Med. 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Analysis of these structures reveals that FTO is comprised of two structural domains separated by an unstructured eight-residue linker. The N-terminal domain (residues 1–322) is competent for catalysis and contains the binding site for the metal cofactor, αKG, and the methylated nucleobase. The C-terminal domain (residues 331–505) does not contact the primary or secondary substrate of FTO but forms an extensive interaction with the N-terminal domain. While the C-terminal domain is required for the correct functioning of FTO (14Han Z. Niu T. Chang J. Lei X. Zhao M. Wang Q. Cheng W. Wang J. Feng Y. Chai J. Crystal structure of the FTO protein reveals basis for its substrate specificity.Nature. 2010; 464: 1205-1209Crossref PubMed Scopus (275) Google Scholar), its exact role in catalysis is still unknown. Here, we investigate the apo form of human FTO and of the isolated N-terminal FTO (nFTO) and C-terminal FTO (cFTO) domains of the enzyme by solution NMR and molecular dynamics (MD) simulations. We show that the interaction between the N- and C-terminal domain is essential to stabilize the structure of the catalytic domain in its active conformation. In addition, by using NMRrelaxation experiments, we establish that FTO is a highly flexible enzyme displaying conformational dynamics both on the picosecond–nanosecond and on the microsecond–millisecond timescale. We then obtained an ensemble representation of FTO conformations in solution by combining residual dipolar couplings (RDCs) with accelerated molecular dynamics (aMD) simulations. Our data indicate that the interface between the FTO domains is more disordered than what observed in the crystal state and undergoes structural fluctuations that result in formation of large surface pockets that can accommodate small-molecule ligands. As the interaction between the N- and C-terminal domain of FTO is crucial for catalysis, these transient pockets can provide the binding site for allosteric inhibitors of the enzyme. This study highlights the ability of solution NMR and MD simulations to characterize structural disorder in proteins and to identify low-population states that are invisible to crystallography and open new possibilities for drug discovery. In this study, we investigated a construct of human FTO in which the first 31 residues are truncated. This construct was shown to retain full enzymatic activity (14Han Z. Niu T. Chang J. Lei X. Zhao M. Wang Q. Cheng W. Wang J. Feng Y. Chai J. Crystal structure of the FTO protein reveals basis for its substrate specificity.Nature. 2010; 464: 1205-1209Crossref PubMed Scopus (275) Google Scholar) and was employed in all crystallographic investigations of FTO. The truncated FTO will be referred to as full-length FTO (as opposed to the isolated nFTO and cFTO domains) in the rest of the article. The 800 MHz 1H–15N transverse relaxation optimized spectroscopy (TROSY) spectrum (22Pervushin K. Riek R. Wider G. Wüthrich K. Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12366Crossref PubMed Scopus (2040) Google Scholar) of 2H,15N-labeled FTO is shown in Figure 1A. Preliminary analysis of the NMR data reveals the presence of 12 signals in the region occupied by the Nε1–Hε1 correlation from the Trp side chain. Since the spectrum is well dispersed and the primary sequence of FTO contains exactly 12 Trp residues, these data suggest that the enzyme is well folded in solution. Of note, we observe the presence of one NH correlation with 1H chemical shift of 2.9 ppm. Assignment of the NMR peaks (see later) reveals that this correlation belongs to Gly312 (Fig. S1). Interestingly, the amide hydrogen of Gly312 is packed against the side chain of Trp270 in the crystal structure of FTO (Fig. 1A, upper left corner). Since the ring current from aromatic groups can result in substantial shift of the NMR resonances, the observation of a large upfield shift for the 1H chemical shift of Gly312 is consistent with the crystal structure of the enzyme. The 1H–15N TROSY spectra acquired for the 2H,15N-labeled nFTO (Fig. 1B) and cFTO (Fig. 1C) domains are of much lower quality compared with the spectrum measured for the full-length enzyme (Fig. 1A). In particular, the NMR peaks are considerably broader and less disperse in the isolated domains than in the full-length protein. In addition, only one Nε1–Hε1 correlation is observed in the Trp side-chain region of the nFTO and cFTO spectra. These data indicate that the isolated nFTO and cFTO are structurally unstable and that the extensive interaction between the N- and C-terminal domain of FTO is absolutely required to fold the enzyme in its functional conformation. Assignment of the 1HN, 15NH, 13Cα, 13Cβ, and 13C′ resonances of FTO was performed using triple resonance methods (23Clore G.M. Gronenborn A.M. Determining the structures of large proteins and protein complexes by NMR.Trends Biotechnol. 1998; 16: 22-34Abstract Full Text PDF PubMed Scopus (223) Google Scholar) with TROSY readout. Selective 15N-labeling of nine amino acids (Arg, Asn, His, Ile, Lys, Leu, Phe, Tyr, and Val) was used to resolve ambiguous assignments (Fig. S2) (24Lacabanne D. Meier B.H. Böckmann A. Selective labeling and unlabeling strategies in protein solid-state NMR spectroscopy.J. Biomol. NMR. 2018; 71: 141-150Crossref PubMed Scopus (31) Google Scholar). About 426 of 449 expected peaks were observed in the 1H–15N TROSY spectrum of FTO (note that the 25 Pro residues are not expected to provide a backbone amide peak). A total of 248 NH correlations were unambiguously assigned (∼55% of the expected peaks). The low assignment rate is due to the large size of the enzyme (54 kDa), its unfavorable relaxation properties, and the inability to produce stable samples of FTO at high concentrations (note that the assignment experiments were measured on samples containing ∼0.3 mM FTO). Nonetheless, the assigned resonances are homogenously distributed on the enzyme structure and cover numerous areas of interest (Figs. 1D and S3). In particular, several assigned amide correlations are localized at the interface between the N- and C-terminal domain of the enzyme, within and surrounding the binding site for the primary and secondary substrates, within the nucleotide recognition loop (residues 213–225) and within unstructured loops that are not observed by crystallography because of the lack of electron density (residues 121–129, 159–188, and 251–263). Of note, the distribution of secondary Cα chemical shifts along the FTO primary sequence is consistent with the secondary structure calculated from the crystal structure of the holo enzyme (Fig. 1E), which provides further evidence that the overall fold of apo FTO in solution resembles the one observed for the holo enzyme in the crystal state. The assigned backbone resonances for FTO were deposited on the BioMagResBank (accession number: 51176) (25Ulrich E.L. Akutsu H. Doreleijers J.F. Harano Y. Ioannidis Y.E. Lin J. Livny M. Mading S. Maziuk D. Miller Z. Nakatani E. Schulte C.F. Tolmie D.E. Kent Wenger R. Yao H. et al.BioMagResBank.Nucleic Acids Res. 2008; 36: D402-D408Crossref PubMed Scopus (1230) Google Scholar). To better investigate the consistency of the crystal structure of holo FTO with the solution structure of the apo enzyme, we have measured backbone amide 1DNH RDC data for apo FTO partially aligned in a dilute liquid crystalline medium of phage pf1 (26Tjandra N. Bax A. Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium.Science. 1997; 278: 1111-1114Crossref PubMed Scopus (1462) Google Scholar). 1DNH RDCs provide information on the orientation of the N–H bond vectors relative to the external magnetic field and are commonly employed to assess the quality of and refine crystallographic structures (27Venditti V. Egner T.K. Clore G.M. Hybrid approaches to structural characterization of conformational ensembles of complex macromolecular systems combining NMR residual dipolar couplings and solution X-ray scattering.Chem. Rev. 2016; 116: 6305-6322Crossref PubMed Scopus (33) Google Scholar). We have measured 1DNH RDC data for 144 and 79 nonoverlapping NMR signals coming from the N- and C-terminal domain of full-length FTO (plus three RDCs coming from the flexible linker), respectively, by using the amide RDCs by TROSY spectroscopy (ARTSY) pulse sequence (Fig. 2D) (28Fitzkee N.C. Bax A. Facile measurement of 1H-15N residual dipolar couplings in larger perdeuterated proteins.J. Biomol. NMR. 2010; 48: 65-70Crossref PubMed Scopus (69) Google Scholar). Interestingly, singular value decomposition fitting of the data coming from secondary structures (80 and 55 RDC values for the N- and C-terminal domain, respectively) to the coordinates of the N- and C-terminal domains of the holo FTO X-ray structure returns R-factors of 60 and 70%, respectively (Fig. 2A). The poor agreement between experimental and back-calculated data indicates that no single orientation of the atomic coordinates in the PDB file of holo FTO (PDB code: 3LFM) can be found that satisfies the experimental RDC data, and, therefore, the crystal structure of holo FTO does not fully capture the behavior of the apo enzyme in solution. Since Alkb enzymes are known to be highly flexible proteins (29Bleijlevens B. Shivarattan T. van den Boom K.S. de Haan A. van der Zwan G. Simpson P.J. Matthews S.J. Changes in protein dynamics of the DNA repair dioxygenase AlkB upon binding of Fe(2+) and 2-oxoglutarate.Biochemistry. 2012; 51: 3334-3341Crossref PubMed Scopus (28) Google Scholar, 30Ergel B. Gill M.L. Brown L. Yu B. Palmer A.G. Hunt J.F. Protein dynamics control the progression and efficiency of the catalytic reaction cycle of the Escherichia coli DNA-repair enzyme AlkB.J. Biol. Chem. 2014; 289: 29584-29601Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 31Purslow J.A. Nguyen T.T. Egner T.K. Dotas R.R. Khatiwada B. Venditti V. Active site breathing of human Alkbh5 revealed by solution NMR and accelerated molecular dynamics.Biophys. J. 2018; 115: 1895-1905Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 32Purslow J.A. Nguyen T.T. Khatiwada B. Singh A. Venditti V. N 6-methyladenosine binding induces a metal-centered rearrangement that activates the human RNA demethylase Alkbh5.Sci. Adv. 2021; 7eabi8215Crossref PubMed Scopus (6) Google Scholar), we ascribe the inconsistency between the crystal structure and solution NMR data to conformational dynamics. To obtain a structural model of apo FTO in solution that would account for conformational dynamics, we have calculated a structural ensemble for the enzyme by coupling the experimental 1DNH RDCs with aMD simulations (31Purslow J.A. Nguyen T.T. Egner T.K. Dotas R.R. Khatiwada B. Venditti V. Active site breathing of human Alkbh5 revealed by solution NMR and accelerated molecular dynamics.Biophys. J. 2018; 115: 1895-1905Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). We have proven this protocol successful in generating MD-derived structural ensembles of dynamical proteins that satisfy solution NMR data (31Purslow J.A. Nguyen T.T. Egner T.K. Dotas R.R. Khatiwada B. Venditti V. Active site breathing of human Alkbh5 revealed by solution NMR and accelerated molecular dynamics.Biophys. J. 2018; 115: 1895-1905Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 32Purslow J.A. Nguyen T.T. Khatiwada B. Singh A. Venditti V. N 6-methyladenosine binding induces a metal-centered rearrangement that activates the human RNA demethylase Alkbh5.Sci. Adv. 2021; 7eabi8215Crossref PubMed Scopus (6) Google Scholar, 33Nguyen T.T. Ghirlando R. Roche J. Venditti V. Structure elucidation of the elusive Enzyme I monomer reveals the molecular mechanisms linking oligomerization and enzymatic activity.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2100298118Crossref Scopus (4) Google Scholar). An ensemble of 39 conformations extracted from the aMD trajectory is required to fulfill the entire set of 226 experimental RDCs (including the data from unstructured regions) (Fig. 2, B and C). The obtained structure ensemble confirms that FTO is a highly flexible enzyme (Fig. 2, E and F). Indeed, regions with high conformational disorder (resulting in large B-factor) are observed at the N terminus (residues 32–45), peripheral loops (residues 173–191, 251–267, 277–282, 345–354, 423–430, and 477–488), and loops located at the interface between the N- and C-terminal domain (residues 84–89, 120–125, and 460–467) (Fig. 2, E and F). It is also important to highlight that an overlay of the crystal structure of holo FTO with the representative structure of the conformational ensemble calculated for apo FTO (i.e., the ensemble member with the lowest backbone rmsd from the average structure calculated over the ensemble) reveals large C⍺ rmsd at several flexible loops located on both the N- and C-terminal domain of the enzyme (Figs. 2G and S4). Although these results may underline ligand-induced conformational changes, it should be noted that these discrepancies between crystal structure and solution structure ensemble could be artifacts deriving from crystal packing. NMR relaxation experiments are a preferred tool for experimental investigations of protein conformational dynamics. In particular, measuring the longitudinal (R1) and transverse (R2) relaxation rates reports on the regions of the protein that are flexible on the picosecond–nanosecond timescale (34Lisi G.P. Loria J.P. Solution NMR spectroscopy for the study of enzyme allostery.Chem. Rev. 2016; 116: 6323-6369Crossref PubMed Scopus (75) Google Scholar). Relaxation dispersion experiments inform on areas of the protein structure that undergo conformational dynamics on the microsecond–millisecond timescale (34Lisi G.P. Loria J.P. Solution NMR spectroscopy for the study of enzyme allostery.Chem. Rev. 2016; 116: 6323-6369Crossref PubMed Scopus (75) Google Scholar, 35Singh A. Purslow J.A. Venditti V. 15N CPMG relaxation dispersion for the investigation of protein conformational dynamics on the micros-ms timescale.J. Vis. Exp. 2021; https://doi.org/10.3791/62395Crossref Scopus (4) Google Scholar). Residue-specific 15N R1 and R2 values were measured at 800 MHz and 30 °C by acquisition of TROSY-detected R1 and R1ρ experiments (36Lakomek N.-A. Ying J. Bax A. Measurement of 1⁵N relaxation rates in perdeuterated proteins by TROSY-based methods.J. Biomol. NMR. 2012; 53: 209-221Crossref PubMed Scopus (118) Google Scholar) on 2H, 15N-labeled FTO and are reported as 15N-R2/R1 ratios in Figure 3, A and C. For a rigid protein, where global rotational tumbling is the only contribution to the picosecond–nanosecond dynamics, the R2/R1 values are expected to be constant throughout the primary sequence and proportional to the rotational correlation time (τc) (37Kay L.E. Torchia D.A. Bax A. Backbone dynamics of proteins as studied by nitrogen-15 inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease.Biochemistry. 1989; 28: 8972-8979Crossref PubMed Scopus (1774) Google Scholar). Instead, the presence of flexible structural elements within the protein (such as long and flexible loops) that locally increase the picosecond–nanosecond dynamics experienced by the backbone amide groups is revealed by a local shift of the R2/R1 ratios toward lower than average values (37Kay L.E. Torchia D.A. Bax A. Backbone dynamics of proteins as studied by nitrogen-15 inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease.Biochemistry. 1989; 28: 8972-8979Crossref PubMed Scopus (1774) Google Scholar). At 30 °C, a globular protein of the size of FTO (54 kDa) is expected to have τc ∼ 29 ns (see the Experimental procedures section), which translates to an 800 MHz 15N-R2/R1 ratio of ∼147. The average 15N-R2/R1 ratio measured for FTO is 107 ± 75, which is consistent with the predicted τc value (Fig. 3A). Interestingly, analysis of the 15N-R2/R1 values versus residue index (Fig. 3, A and C) reveals the presence of several residues with a lower than average 15N-R2/R1. These residues are localized at the N- and C-terminal ends of the protein (residues 34–36 and 502–504, respectively) and within the peripheral loops displaying large B-factors in the RDC/aMD conformational ensembles (residues 164–193, 248–265, 279–285, 349–356, and 424–425) (compare Figs. 2E and 3C). Relaxation dispersion data (800 MHz 15N) were measured on 2H, 15N-labeled FTO at 30 and 15 °C using the Carr–Purcell–Meinboom–Gill (CPMG) experiment (35Singh A. Purslow J.A. Venditti V. 15N CPMG relaxation dispersion for the investigation of protein conformational dynamics on the micros-ms timescale.J. Vis. Exp. 2021; https://doi.org/10.3791/62395Crossref Scopus (4) Google Scholar, 38Mittermaier A. Kay L.E. New tools provide new insights in NMR studies of protein dynamics.Science. 2006; 312: 224-228Crossref PubMed Scopus (626) Google Scholar). Significant relaxation dispersion was observed for the backbone amides of 16 residues (Figs. 3, B and D and S5). Of note, six of these residues (I193, Y199, Y214, L215, V228, and V345) do not fall in a well-defined area of FTO but are scattered within the protein structure (Fig. 3D). We ascribe the relaxation dispersions observed at these residues to local microsecond–millisecond timescale structural fluctuations that affect the 15N chemical shift of a single amide group (i.e., formation/disruption of a hydrogen bond and/or rearrangement of a nearby side chain). On the other hand, the remaining 10 relaxation disper
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