An α-ketoglutarate conformational switch controls iron accessibility, activation, and substrate selection of the human FTO protein.

The fat mass and obesity-associated fatso (FTO) protein is a member of the Alkb family of dioxygenases and catalyzes oxidative demethylation of N6-methyladenosine (m6A), N1-methyladenosine (m1A), 3-methylthymine (m3T), and 3-methyluracil (m3U) in single-stranded nucleic acids. It is well established that the catalytic activity of FTO proceeds via two coupled reactions. The first reaction involves decarboxylation of alpha-ketoglutarate (αKG) and formation of an oxyferryl species. In the second reaction, the oxyferryl intermediate oxidizes the methylated nucleic acid to reestablish Fe(II) and the canonical base. However, it remains unclear how binding of the nucleic acid activates the αKG decarboxylation reaction and why FTO demethylates different methyl modifications at different rates. Here, we investigate the interaction of FTO with 5-mer DNA oligos incorporating the m6A, m1A, or m3T modifications using solution NMR, molecular dynamics (MD) simulations, and enzymatic assays. We show that binding of the nucleic acid to FTO activates a two-state conformational equilibrium in the αKG cosubstrate that modulates the O2 accessibility of the Fe(II) catalyst. Notably, the substrates that provide better stabilization to the αKG conformation in which Fe(II) is exposed to O2 are demethylated more efficiently by FTO. These results indicate that i) binding of the methylated nucleic acid is required to expose the catalytic metal to O2 and activate the αKG decarboxylation reaction, and ii) the measured turnover of the demethylation reaction (which is an ensemble average over the entire sample) depends on the ability of the methylated base to favor the Fe(II) state accessible to O2.

Elucidation of the Mechanisms of Inter-domain Coupling in the Monomeric State of Enzyme I by High-pressure NMR.

The catalytic cycle of Enzyme I (EI), a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate, is characterized by a series of local and global conformational rearrangements. This multistep process includes a monomer-to-dimer transition, followed by an open-to-closed rearrangement of the dimeric complex upon PEP binding. In the present study, we investigate the thermodynamics of EI dimerization using a range of high-pressure solution NMR techniques complemented by SAXS experiments. 1H-15N TROSY and 1H-13C methyl TROSY NMR spectra combined with 15N relaxation measurements revealed that a native-like engineered variant of full-length EI fully dissociates into stable monomeric state above 1.5 kbar. Conformational ensembles of EI monomeric state were generated via a recently developed protocol combining coarse-grained molecular simulations with experimental backbone residual dipolar coupling measurements. Analysis of the structural ensembles provided detailed insights into the molecular mechanisms driving formation of the catalytically competent dimeric state, and reveals that each step of EI catalytical cycle is associated with a significant reduction in either inter- or intra-domain conformational entropy. Altogether, this study completes a large body work conducted by our group on EI and establishes a comprehensive structural and dynamical description of the catalytic cycle of this prototypical multidomain, oligomeric enzyme.

Hydrogen spillover and substrate-support hydrogen bonding mediate hydrogenation of phenol catalyzed by palladium on reducible metal oxides.

Substrate-support interactions play an important role in the catalytic hydrogenation of phenolic compounds by ceria-supported palladium (Pd/CeO2). Here, we combine surface contrast solution NMR methods and reaction kinetic assays to investigate the role of substrate-support interactions in phenol (PhOH) hydrogenation catalyzed by titania-supported palladium (Pd/TiO2). We show that PhOH adsorbs on the catalyst via a weak hydrogen-bonding interaction between the -OH group of the substrate and one oxygen atom on the support. Interestingly, we observe that the addition of 20 mM inorganic phosphate results in a ∼2-fold destabilization of the PhOH-support interaction and a corresponding ∼2-fold inhibition of the catalytic reaction, suggesting an active role of the PhOH-TiO2 hydrogen bond in catalysis. A comparison of the data measured here with the results previously reported for a Pd/CeO2 catalyst indicates that the efficiency of the Pd-supported catalysts is correlated to the amount of PhOH hydrogen bonded to the metal oxide support. Since CeO2 and TiO2 have similar ability to uptake activated hydrogen from a noble metal site, these data suggest that hydrogen spillover is the main mechanism by which Pd-activated hydrogens are shuttled to the PhOH adsorbed on the surface of the support. Consistent with this hypothesis, Pd supported on a non-reducible metal oxide (silica) displays negligible hydrogenation activity. Therefore, we conclude that basic and reducible metal oxides are active supports for the efficient hydrogenation of phenolic compounds due to their ability to hydrogen bond to the substrate and mediate the addition of the activated hydrogens to the adsorbed aromatic ring.

Temperature sensitive contact modes allosterically gate TRPV3.

TRPV Ion channels are sophisticated molecular sensors designed to respond to distinct temperature thresholds. The recent surge in cryo-EM structures has provided numerous insights into the structural rearrangements accompanying their opening and closing; however, the molecular mechanisms by which TRPV channels establish precise and robust temperature sensing remain elusive. In this work we employ molecular simulations, multi-ensemble contact analysis, graph theory, and machine learning techniques to reveal the temperature-sensitive residue-residue interactions driving allostery in TRPV3. We find that groups of residues exhibiting similar temperature-dependent contact frequency profiles cluster at specific regions of the channel. The dominant mode clusters on the ankyrin repeat domain and displays a linear melting trend while others display non-linear trends. These modes describe the residue-level temperature response patterns that underlie the channel's functional dynamics. With network analysis, we find that the community structure of the channel changes with temperature. And that a network of high centrality contacts connects distant regions of the protomer to the gate, serving as a means for the temperature-sensitive contact modes to allosterically regulate channel gating. Using a random forest model, we show that the contact states of specific temperature-sensitive modes are indeed predictive of the channel gate's state. Supporting the physical validity of these modes and networks are several residues identified with our analyses that are reported in literature to be functionally critical. Our results offer high resolution insight into thermo-TRP channel function and demonstrate the utility of temperature-sensitive contact analysis.

Solution Structure Ensembles of the Open and Closed Forms of the ∼130 kDa Enzyme I via AlphaFold Modeling, Coarse Grained Simulations, and NMR.

Large-scale interdomain rearrangements are essential to protein function, governing the activity of large enzymes and molecular machineries. Yet, obtaining an atomic-resolution understanding of how the relative domain positioning is affected by external stimuli is a hard task in modern structural biology. Here, we show that combining structural modeling by AlphaFold2 with coarse-grained molecular dynamics simulations and NMR residual dipolar coupling data is sufficient to characterize the spatial domain organization of bacterial enzyme I (EI), a ∼130 kDa multidomain oligomeric protein that undergoes large-scale conformational changes during its catalytic cycle. In particular, we solve conformational ensembles for EI at two different experimental temperatures and demonstrate that a lower temperature favors sampling of the catalytically competent closed state of the enzyme. These results suggest a role for conformational entropy in the activation of EI and demonstrate the ability of our protocol to detect and characterize the effect of external stimuli (such as mutations, ligand binding, and post-translational modifications) on the interdomain organization of multidomain proteins. We expect the ensemble refinement protocol described here to be easily transferrable to the investigation of the structure and dynamics of other uncharted multidomain systems and have assembled a Google Colab page (https://potoyangroup.github.io/Seq2Ensemble/) to facilitate implementation of the presented methodology elsewhere.

Protein Conformational Dynamics Underlie Selective Recognition of Thermophilic over Mesophilic Enzyme I by a Substrate Analogue.

Substrate selectivity is an important preventive measure to decrease the possibility of cross interactions between enzymes and metabolites that share structural similarities. In addition, understanding the mechanisms that determine selectivity towards a particular substrate increases the knowledge base for designing specific inhibitors for target enzymes. Here, we combine NMR, molecular dynamics (MD) simulations, and protein engineering to investigate how two substrate analogues, allylicphosphonate (cPEP) and sulfoenolpyruvate (SEP), recognize the mesophilic (eEIC) and thermophilic (tEIC) homologues of the receptor domain of bacterial Enzyme I, which has been proposed as a target for antimicrobial research. Chemical Shift Perturbation (CSP) experiments show that cPEP and SEP recognize tEIC over the mesophilic homologue. Combined Principal Component Analysis of half-microsecond-long MD simulations reveals that incomplete quenching of a breathing motion in the eEIC-ligand complex destabilizes the interaction and makes the investigated substrate analogues selective toward the thermophilic enzyme. Our results indicate that residual protein motions need to be considered carefully when optimizing small molecule inhibitors of EI. In general, our work demonstrates that protein conformational dynamics can be exploited in the rational design and optimization of inhibitors with subfamily selectivity.

Temperature-Sensitive Contact Modes Allosterically Gate TRPV3.

TRPV Ion channels are sophisticated molecular sensors designed to respond to distinct temperature thresholds. The recent surge in cryo-EM structures has provided numerous insights into the structural rearrangements accompanying their opening and closing; however, the molecular mechanisms by which TRPV channels establish precise and robust temperature sensing remain elusive. In this work we employ molecular simulations, multi-ensemble contact analysis, graph theory, and machine learning techniques to reveal the temperature-sensitive residue-residue interactions driving allostery in TRPV3. We find that groups of residues exhibiting similar temperature-dependent contact frequency profiles cluster at specific regions of the channel. The dominant mode clusters on the ankyrin repeat domain and displays a linear melting trend while others display non-linear trends. These modes describe the residue-level temperature response patterns that underlie the channel's functional dynamics. With network analysis, we find that the community structure of the channel changes with temperature. And that a network of high centrality contacts connects distant regions of the protomer to the gate, serving as a means for the temperature-sensitive contact modes to allosterically regulate channel gating. Using a random forest model, we show that the contact states of specific temperature-sensitive modes are indeed predictive of the channel gate's state. Supporting the physical validity of these modes and networks are several residues identified with our analyses that are reported in literature to be functionally critical. Our results offer high resolution insight into thermo-TRP channel function and demonstrate the utility of temperature-sensitive contact analysis.

Temperature-sensitive contacts in disordered loops tune enzyme I activity.

Homologous enzymes with identical folds often exhibit different thermal and kinetic behaviors. Understanding how an enzyme sequence encodes catalytic activity at functionally optimal temperatures is a fundamental problem in biophysics. Recently it was shown that the residues that tune catalytic activities of thermophilic/mesophilic variants of the C-terminal domain of bacterial enzyme I (EIC) are largely localized within disordered loops, offering a model system with which to investigate this phenomenon. In this work, we use molecular dynamics simulations and mutagenesis experiments to reveal a mechanism of sequence-dependent activity tuning of EIC homologs. We find that a network of contacts in the catalytic loops is particularly sensitive to changes in temperature, with some contacts exhibiting distinct linear or nonlinear temperature-dependent trends. Moreover, these trends define structurally clustered dynamical modes and can distinguish regions that tend toward order or disorder at higher temperatures. Assaying several thermophilic EIC mutants, we show that complementary mesophilic mutations to the most temperature-sensitive positions exhibit the most enhanced activity, while mutations to relatively temperature insensitive positions exhibit the least enhanced activities. These results provide a mechanistic explanation of sequence-dependent temperature tuning and offer a computational method for rational enzyme modification.

Solution NMR methods for structural and thermodynamic investigation of nanoparticle adsorption equilibria.

Characterization of dynamic processes occurring at the nanoparticle (NP) surface is crucial for developing new and more efficient NP catalysts and materials. Thus, a vast amount of research has been dedicated to developing techniques to characterize sorption equilibria. Over recent years, solution NMR spectroscopy has emerged as a preferred tool for investigating ligand-NP interactions. Indeed, due to its ability to probe exchange dynamics over a wide range of timescales with atomic resolution, solution NMR can provide structural, kinetic, and thermodynamic information on sorption equilibria involving multiple adsorbed species and intermediate states. In this contribution, we review solution NMR methods for characterizing ligand-NP interactions, and provide examples of practical applications using these methods as standalone techniques. In addition, we illustrate how the integrated analysis of several NMR datasets was employed to elucidate the role played by support-substrate interactions in mediating the phenol hydrogenation reaction catalyzed by ceria-supported Pd nanoparticles.

Solution structure ensemble of human obesity-associated protein FTO reveals druggable surface pockets at the interface between the N- and C-terminal domain.

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.

N 6-methyladenosine binding induces a metal-centered rearrangement that activates the human RNA demethylase Alkbh5.

Alkbh5 catalyzes demethylation of the N 6-methyladenosine (m6A), an epigenetic mark that controls several physiological processes including carcinogenesis and stem cell differentiation. The activity of Alkbh5 comprises two coupled reactions. The first reaction involves decarboxylation of α-ketoglutarate (αKG) and formation of a Fe4+═O species. This oxyferryl intermediate oxidizes the m6A to reestablish the canonical base. Despite coupling between the two reactions being required for the correct Alkbh5 functioning, the mechanisms linking dioxygen activation to m6A binding are not fully understood. Here, we use solution NMR to investigate the structure and dynamics of apo and holo Alkbh5. We show that binding of m6A to Alkbh5 induces a metal-centered rearrangement of αKG that increases the exposed area of the metal, making it available for binding O2 Our study reveals the molecular mechanisms underlying activation of Alkbh5, therefore opening new perspectives for the design of novel strategies to control gene expression and cancer progression.

A Single Point Mutation Controls the Rate of Interconversion Between the g + and g - Rotamers of the Histidine 189 χ2 Angle That Activates Bacterial Enzyme I for Catalysis.

Enzyme I (EI) of the bacterial phosphotransferase system (PTS) is a master regulator of bacterial metabolism and a promising target for development of a new class of broad-spectrum antibiotics. The catalytic activity of EI is mediated by several intradomain, interdomain, and intersubunit conformational equilibria. Therefore, in addition to its relevance as a drug target, EI is also a good model for investigating the dynamics/function relationship in multidomain, oligomeric proteins. Here, we use solution NMR and protein design to investigate how the conformational dynamics occurring within the N-terminal domain (EIN) affect the activity of EI. We show that the rotameric g + -to-g - transition of the active site residue His189 χ2 angle is decoupled from the state A-to-state B transition that describes a ∼90° rigid-body rearrangement of the EIN subdomains upon transition of the full-length enzyme to its catalytically competent closed form. In addition, we engineered EIN constructs with modulated conformational dynamics by hybridizing EIN from mesophilic and thermophilic species, and used these chimeras to assess the effect of increased or decreased active site flexibility on the enzymatic activity of EI. Our results indicate that the rate of the autophosphorylation reaction catalyzed by EI is independent from the kinetics of the g + -to-g - rotameric transition that exposes the phosphorylation site on EIN to the incoming phosphoryl group. In addition, our work provides an example of how engineering of hybrid mesophilic/thermophilic chimeras can assist investigations of the dynamics/function relationship in proteins, therefore opening new possibilities in biophysics.

Structure elucidation of the elusive Enzyme I monomer reveals the molecular mechanisms linking oligomerization and enzymatic activity.

Enzyme I (EI) is a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate. This reaction initiates a five-step phosphorylation cascade in the bacterial phosphotransferase (PTS) transduction pathway. Under physiological conditions, EI exists in an equilibrium between a functional dimer and an inactive monomer. The monomer-dimer equilibrium is a crucial factor regulating EI activity and the phosphorylation state of the overall PTS. Experimental studies of EI's monomeric state have yet been hampered by the dimer's high thermodynamic stability, which prevents its characterization by standard structural techniques. In this study, we modified the dimerization domain of EI (EIC) by mutating three amino acids involved in the formation of intersubunit salt bridges. The engineered variant forms an active dimer in solution that can bind and hydrolyze PEP. Using hydrostatic pressure as an additional perturbation, we were then able to study the complete dissociation of the variant from 1 bar to 2.5 kbar in the absence and the presence of EI natural ligands. Backbone residual dipolar couplings collected under high-pressure conditions allowed us to determine the conformational ensemble of the isolated EIC monomeric state in solution. Our calculations reveal that three catalytic loops near the dimerization interface become unstructured upon monomerization, preventing the monomeric enzyme from binding its natural substrate. This study provides an atomic-level characterization of EI's monomeric state and highlights the role of the catalytic loops as allosteric connectors controlling both the activity and oligomerization of the enzyme.

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the µs-ms Timescale.

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important biological processes. Understanding how the balance between structure and dynamics governs biological function is a new frontier in modern structural biology and has ignited several technical and methodological developments. Among these, CPMG relaxation dispersion solution NMR methods provide unique, atomic-resolution information on the structure, kinetics, and thermodynamics of protein conformational equilibria occurring on the µs-ms timescale. Here, the study presents detailed protocols for acquisition and analysis of a 15N relaxation dispersion experiment. As an example, the pipeline for the analysis of the µs-ms dynamics in the C-terminal domain of bacteria Enzyme I is shown.

An organogel library for solution NMR analysis of nanoparticle suspensions in non-aqueous samples.

Surface contrast solution NMR methods (scNMR) are emerging as powerful tools to investigate the adsorption of small molecule ligands to the surface of nanoparticles (NP), returning fundamental insight into the kinetics and thermodynamics of sorption, as well as structural information on the adsorbed species. A prerequisite for the acquisition of high quality solution NMR data is the preparation of homogeneous and stable samples that return consistent NMR spectra and allow extensive signal averaging. Unfortunately, this condition does not apply to NMR samples containing NPs that often show a tendency to sediment and accumulate at the bottom of the NMR tube over the course of the experiment. We have recently shown that preparing NMR samples in an agarose gel matrix inhibits sedimentation and allows the characterization of small molecule-NP interactions by scNMR. Unfortunately, as the agarose gel only forms in aqueous solution, this sample preparation method cannot be used to stabilize NP suspensions in a non-aqueous environment. Here, we introduce a library of 48 organogels, based on low molecular-mass organic gelators (LMOGs), to prepare NMR samples of small molecule/NP systems in a wide range of organic solvents. In addition, we present a simple method that takes advantage of 1H transverse relaxation (1H-R2) measurements to screen the library and identify the best gelator to characterize the small molecule-NP interaction of interest in the solvent of choice. We expect the results of this study will enable the preparation of homogeneous and stable samples of NPs in non-aqueous environments, therefore dramatically increasing the applicability of scNMR to the characterization of heterogeneous interactions and to the investigation of the role played by solvent molecules in regulating the kinetics and thermodynamics of sorption.

Weak binding to the A2RE RNA rigidifies hnRNPA2 RRMs and reduces liquid-liquid phase separation and aggregation.

hnRNPA2 is a major component of mRNA transport granules in oligodendrocytes and neurons. However, the structural details of how hnRNPA2 binds the A2 recognition element (A2RE) and if this sequence stimulates granule formation by enhancing phase separation of hnRNPA2 has not yet been studied. Using solution NMR and biophysical studies, we find that each of the two individual RRMs retain the domain structure observed in complex with RNA but are not rigidly confined (i.e. they move independently) in solution in the absence of RNA. hnRNPA2 RRMs bind the minimal rA2RE11 weakly but at least, and most likely, two hnRNPA2 molecules are able to simultaneously bind the longer 21mer myelin basic protein A2RE. Upon binding of the RNA, NMR chemical shift deviations are observed in both RRMs, suggesting both play a role in binding the A2RE11. Interestingly, addition of short A2RE RNAs or longer RNAs containing this sequence completely prevents in vitro phase separation of full-length hnRNPA2 and aggregation of the disease-associated mutants. These findings suggest that RRM interactions with specific recognition sequences alone do not account for nucleating granule formation, consistent with models where multivalent protein:RNA and protein:protein contacts form across many sites in granule proteins and long RNA transcripts.

An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screening.

Enzyme I (EI), which is the key enzyme to activate the bacterial phosphotransferase system, plays an important role in the regulation of several metabolic pathways and controls the biology of bacterial cells at multiple levels. The conservation and ubiquity of EI among different types of bacteria makes the enzyme a potential target for antimicrobial research. Here, we use NMR-based fragment screening to identify novel inhibitors of EI. We identify three molecular fragments that allosterically inhibit the phosphoryl transfer reaction catalyzed by EI by interacting with the enzyme at a surface pocket located more than 10 Å away from the substrate binding site. Interestingly, although the three molecules share the same binding pocket, we observe that two of the discovered EI ligands act as competitive inhibitors while the third ligand acts as a mixed inhibitor. Characterization of the EI-inhibitor complexes by NMR and Molecular Dynamics simulations reveals key interactions that perturb the fold of the active site and provides structural foundation for the different inhibitory activity of the identified molecular fragments. In particular, we show that contacts between the inhibitor and the side-chain of V292 are crucial to destabilize binding of the substrate to EI. In contrast, mixed inhibition is caused by additional contacts between the inhibitor and ⍺-helix 2 that perturb the active site structure and turnover in an allosteric manner. We expect our results to provide the basis for the development of second generation allosteric inhibitors of increased potency and to suggest novel molecular strategies to combat drug-resistant infections.

Hybrid Thermophilic/Mesophilic Enzymes Reveal a Role for Conformational Disorder in Regulation of Bacterial Enzyme I.

Conformational disorder is emerging as an important feature of biopolymers, regulating a vast array of cellular functions, including signaling, phase separation, and enzyme catalysis. Here we combine NMR, crystallography, computer simulations, protein engineering, and functional assays to investigate the role played by conformational heterogeneity in determining the activity of the C-terminal domain of bacterial Enzyme I (EIC). In particular, we design chimeric proteins by hybridizing EIC from thermophilic and mesophilic organisms, and we characterize the resulting constructs for structure, dynamics, and biological function. We show that EIC exists as a mixture of active and inactive conformations and that functional regulation is achieved by tuning the thermodynamic balance between active and inactive states. Interestingly, we also present a hybrid thermophilic/mesophilic enzyme that is thermostable and more active than the wild-type thermophilic enzyme, suggesting that hybridizing thermophilic and mesophilic proteins is a valid strategy to engineer thermostable enzymes with significant low-temperature activity.

NMR Methods for Structural Characterization of Protein-Protein Complexes.

Protein-protein interactions and the complexes thus formed are critical elements in a wide variety of cellular events that require an atomic-level description to understand them in detail. Such complexes typically constitute challenging systems to characterize and drive the development of innovative biophysical methods. NMR spectroscopy techniques can be applied to extract atomic resolution information on the binding interfaces, intermolecular affinity, and binding-induced conformational changes in protein-protein complexes formed in solution, in the cell membrane, and in large macromolecular assemblies. Here we discuss experimental techniques for the characterization of protein-protein complexes in both solution NMR and solid-state NMR spectroscopy. The approaches include solvent paramagnetic relaxation enhancement and chemical shift perturbations (CSPs) for the identification of binding interfaces, and the application of intermolecular nuclear Overhauser effect spectroscopy and residual dipolar couplings to obtain structural constraints of protein-protein complexes in solution. Complementary methods in solid-state NMR are described, with emphasis on the versatility provided by heteronuclear dipolar recoupling to extract intermolecular constraints in differentially labeled protein complexes. The methods described are of particular relevance to the analysis of membrane proteins, such as those involved in signal transduction pathways, since they can potentially be characterized by both solution and solid-state NMR techniques, and thus outline key developments in this frontier of structural biology.