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.

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.

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.

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.

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.

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.

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.

Hydrogel-based transparent soils for root phenotyping in vivo.

Root phenotypes are increasingly explored as predictors of crop performance but are still challenging to characterize. Media that mimic field conditions (e.g., soil, sand) are opaque to most forms of radiation, while transparent media do not provide field-relevant growing conditions and phenotypes. We describe here a "transparent soil" formed by the spherification of hydrogels of biopolymers. It is specifically designed to support root growth in the presence of air, water, and nutrients, and allows the time-resolved phenotyping of roots in vivo by both photography and microscopy. The roots developed by soybean plants in this medium are significantly more similar to those developed in real soil than those developed in hydroponic conditions and do not show signs of hypoxia. Lastly, we show that the granular nature and tunable properties of these hydrogel beads can be leveraged to investigate the response of roots to gradients in water availability and soil stiffness.

N-terminal fusion of the N-terminal domain of bacterial enzyme I facilitates recombinant expression and purification of the human RNA demethylases FTO and Alkbh5.

Various fusion tags are commonly employed to increase the heterologous expression and solubility of aggregation-prone proteins within Escherichia coli. Herein, we present a protocol for efficient recombinant expression and purification of the human RNA demethylases Alkbh5 and FTO. Our method incorporates a novel fusion tag (the N-terminal domain of bacterial enzyme I, EIN) that dramatically increases the solubility of its fusion partner and is promptly removed upon digestion with a protease. The presented protocol allows for the production of mg amounts of Alkbh5 and FTO in 1L of both rich and minimal media. We developed a liquid chromatography-mass spectrometry (LC-MS)-based assay to confirm that both proteins are enzymatically active. Furthermore, the LC-MS method developed here is applicable to other members of the AlkB family of Fe(II)/α-ketoglutarate-dependent dioxygenases. The superior protein yield, afforded by our expression and purification method, will facilitate biochemical investigations into the biological function of the human RNA demethylases and endorse employment of EIN as a broadly applicable fusion tag for recombinant expression projects.

The oligomerization state of bacterial enzyme I (EI) determines EI's allosteric stimulation or competitive inhibition by α-ketoglutarate.

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by phosphorylation of enzyme I (EI) by phosphoenolpyruvate (PEP). The EI phosphorylation state determines the phosphorylation states of all other PTS components and is thought to play a central role in the regulation of several metabolic pathways and to control the biology of bacterial cells at multiple levels, for example, affecting virulence and biofilm formation. Given the pivotal role of EI in bacterial metabolism, an improved understanding of the mechanisms controlling its activity could inform future strategies for bioengineering and antimicrobial design. Here, we report an enzymatic assay, based on Selective Optimized Flip Angle Short Transient (SOFAST) NMR experiments, to investigate the effect of the small-molecule metabolite α-ketoglutarate (αKG) on the kinetics of the EI-catalyzed phosphoryl transfer reaction. We show that at experimental conditions favoring the monomeric form of EI, αKG promotes dimerization and acts as an allosteric stimulator of the enzyme. However, when the oligomerization state of EI is shifted toward the dimeric species, αKG functions as a competitive inhibitor of EI. We developed a kinetic model that fully accounted for the experimental data and indicated that bacterial cells might use the observed interplay between allosteric stimulation and competitive inhibition of EI by αKG to respond to physiological fluctuations in the intracellular environment. We expect that the mechanism for regulating EI activity revealed here is common to several other oligomeric enzymes.

Active Site Breathing of Human Alkbh5 Revealed by Solution NMR and Accelerated Molecular Dynamics.

AlkB homolog 5 (Alkbh5) is one of nine members of the AlkB family, which are nonheme Fe2+/α-ketoglutarate-dependent dioxygenases that catalyze the oxidative demethylation of modified nucleotides and amino acids. Alkbh5 is highly selective for the N6-methyladenosine modification, an epigenetic mark that has spawned significant biological and pharmacological interest because of its involvement in important physiological processes, such as carcinogenesis and stem cell differentiation. Herein, we investigate the structure and dynamics of human Alkbh5 in solution. By using 15N and 13Cmethyl relaxation dispersion and 15N-R1 and R1ρ NMR experiments, we show that the active site of apo Alkbh5 experiences conformational dynamics on multiple timescales. Consistent with this observation, backbone amide residual dipolar couplings measured for Alkbh5 in phage pf1 are inconsistent with the static crystal structure of the enzyme. We developed a simple approach that combines residual dipolar coupling data and accelerated molecular dynamics simulations to calculate a conformational ensemble of Alkbh5 that is fully consistent with the experimental NMR data. Our structural model reveals that Alkbh5 is more disordered in solution than what is observed in the crystal state and undergoes breathing motions that expand the active site and allow access to α-ketoglutarate. Disordered-to-ordered conformational changes induced by sequential substrate/cofactor binding events have been often invoked to interpret biochemical data on the activity and specificity of AlkB proteins. The structural ensemble reported in this work provides the first atomic-resolution model of an AlkB protein in its disordered conformational state to our knowledge.

Hybrid Approaches to Structural Characterization of Conformational Ensembles of Complex Macromolecular Systems Combining NMR Residual Dipolar Couplings and Solution X-ray Scattering.

Solving structures or structural ensembles of large macromolecular systems in solution poses a challenging problem. While NMR provides structural information at atomic resolution, increased spectral complexity, chemical shift overlap, and short transverse relaxation times (associated with slow tumbling) render application of the usual techniques that have been so successful for medium sized systems (<50 kDa) difficult. Solution X-ray scattering, on the other hand, is not limited by molecular weight but only provides low resolution structural information related to the overall shape and size of the system under investigation. Here we review how combining atomic resolution structures of smaller domains with sparse experimental data afforded by NMR residual dipolar couplings (which yield both orientational and shape information) and solution X-ray scattering data in rigid-body simulated annealing calculations provides a powerful approach for investigating the structural aspects of conformational dynamics in large multidomain proteins. The application of this hybrid methodology is illustrated for the 128 kDa dimer of bacterial Enzyme I which exists in a variety of open and closed states that are sampled at various points in the catalytic cycles, and for the capsid protein of the human immunodeficiency virus.

Dynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering.

Enzyme I (EI) is the first component in the bacterial phosphotransferase system, a signal transduction pathway in which phosphoryl transfer through a series of bimolecular protein-protein interactions is coupled to sugar transport across the membrane. EI is a multidomain, 128-kDa homodimer that has been shown to exist in two conformational states related to one another by two large (50-90°) rigid body domain reorientations. The open conformation of apo EI allows phosphoryl transfer from His189 located in the N-terminal domain α/ß (EIN(α/ß)) subdomain to the downstream protein partner bound to the EIN(α) subdomain. The closed conformation, observed in a trapped phosphoryl transfer intermediate, brings the EIN(α/ß) subdomain into close proximity to the C-terminal dimerization domain (EIC), thereby permitting in-line phosphoryl transfer from phosphoenolpyruvate (PEP) bound to EIC to His189. Here, we investigate the solution conformation of a complex of an active site mutant of EI (H189A) with PEP. Simulated annealing refinement driven simultaneously by solution small angle X-ray scattering and NMR residual dipolar coupling data demonstrates unambiguously that the EI(H189A)-PEP complex exists in a dynamic equilibrium between two approximately equally populated conformational states, one corresponding to the closed structure and the other to a partially closed species. The latter likely represents an intermediate in the open-to-closed transition.

Structure, dynamics and biophysics of the cytoplasmic protein-protein complexes of the bacterial phosphoenolpyruvate: sugar phosphotransferase system.

The bacterial phosphotransferase system (PTS) couples phosphoryl transfer, via a series of bimolecular protein-protein interactions, to sugar transport across the membrane. The multitude of complexes in the PTS provides a paradigm for studying protein interactions, and for understanding how the same binding surface can specifically recognize a diverse array of targets. Fifteen years of work aimed at solving the solution structures of all soluble protein-protein complexes of the PTS has served as a test bed for developing NMR and integrated hybrid approaches to study larger complexes in solution and to probe transient, spectroscopically invisible states, including encounter complexes. We review these approaches, highlighting the problems that can be tackled with these methods, and summarize the current findings on protein interactions.

Mechanistic Insight into Nanoparticle Surface Adsorption by Solution NMR Spectroscopy in an Aqueous Gel.

Engineering nanoparticle (NP) functions at the molecular level requires a detailed understanding of the dynamic processes occurring at the NP surface. Herein we show that a combination of dark-state exchange saturation transfer (DEST) and relaxation dispersion (RD) NMR experiments on gel-stabilized NP samples enables the accurate determination of the kinetics and thermodynamics of adsorption. We used the former approach to describe the interaction of cholic acid (CA) and phenol (PhOH) with ceria NPs with a diameter of approximately 200 nm. Whereas CA formed weak interactions with the NPs, PhOH was tightly bound to the NP surface. Interestingly, we found that the adsorption of PhOH proceeds via an intermediate, weakly bound state in which the small molecule has residual degrees of rotational diffusion. We believe the use of aqueous gels for stabilizing NP samples will increase the applicability of solution NMR methods to the characterization of nanomaterials.

A NMR experiment for simultaneous correlations of valine and leucine/isoleucine methyls with carbonyl chemical shifts in proteins.

A methyl-detected 'out-and-back' NMR experiment for obtaining simultaneous correlations of methyl resonances of valine and isoleucine/leucine residues with backbone carbonyl chemical shifts, SIM-HMCM(CGCBCA)CO, is described. The developed pulse-scheme serves the purpose of convenience in recording a single data set for all Ile(δ1), Leu(δ) and Val(γ) (ILV) methyl positions instead of acquiring two separate spectra selective for valine or leucine/isoleucine residues. The SIM-HMCM(CGCBCA)CO experiment can be used for ILV methyl assignments in moderately sized protein systems (up to ~100 kDa) where the backbone chemical shifts of (13)C(α), (13)Cß and (13)CO are known from prior NMR studies and where some losses in sensitivity can be tolerated for the sake of an overall reduction in NMR acquisition time.

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.

Conformational selection and substrate binding regulate the monomer/dimer equilibrium of the C-terminal domain of Escherichia coli enzyme I.

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by the binding of phosphoenolpyruvate (PEP) to the C-terminal domain (EIC) of enzyme I (EI), a highly conserved protein that is common to all sugar branches of the PTS. EIC exists in a dynamic monomer/dimer equilibrium that is modulated by ligand binding and is thought to regulate the overall PTS. Isolation of EIC has proven challenging, and conformational dynamics within the EIC domain during the catalytic cycle are still largely unknown. Here, we present a robust protocol for expression and purification of recombinant EIC from Escherichia coli and show that isolated EIC is capable of hydrolyzing PEP. NMR analysis and residual dipolar coupling measurements indicate that the isolated EIC domain in solution adopts a stable tertiary fold and quaternary structure that is consistent with previously reported crystallographic data. NMR relaxation dispersion measurements indicate that residues around the PEP binding site and in the ß3α3 turn (residues 333-366), which is located at the dimer interface, undergo a rapid transition on the sub-millisecond time scale (with an exchange rate constant of ∼1500 s(-1)) between major open (∼97%) and minor closed (∼3%) conformations. Upon PEP binding, the ß3α3 turn is effectively locked in the closed state by the formation of salt bridges between the phosphate group of PEP and the side chains of Lys(340) and Arg(358), thereby stabilizing the dimer.

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.

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.