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.

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.

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.

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.

Ring opening of acylated ß-d-arabinofuranose 1,2,5-orthobenzoates with nucleophiles allows access to novel selectively-protected arabinofuranose building blocks.

ß-d-Arabinofuranose 1,2,5-orthobenzoates with 3-O-acetyl, 3-O-benzoyl, and 3-O-chloroacetyl groups were prepared in an efficient manner starting from readily available crystalline methyl 2,3,5-tri-O-benzoyl-α-d-arabinofuranoside, and ring-opening reactions of these compounds with O- and S-nucleophiles were studied. Optimized conditions leading to the formation of the respective monosaccharide adducts (up to 96% isolated yields) and to α-(1→5)-linked disaccharide thioglycosides with 5'-OH unprotected (up to 30% isolated yields) were found. Basing on these results, a novel approach for effective differentiation of 3,5-diol system and 2-hydroxy group in arabinofuranose thioglycosides was proposed. The selectively protected derivatives prepared are valuable building blocks for the assembly of linear and branched oligoarabinofuranosides.