Ligand 1 H NMR Chemical Shifts as Accurate Reporters for Protein-Ligand Binding Interfaces in Solution.

The availability of high-resolution 3D structural information is crucial for investigating guest-host systems across a wide range of fields. In the context of drug discovery, the information is routinely used to establish and validate structure-activity relationships, grow initial hits from screening campaigns, and to guide molecular docking. For the generation of protein-ligand complex structural information, X-ray crystallography is the experimental method of choice, however, with limited information on protein flexibility. An experimentally verified structural model of the binding interface in the native solution-state would support medicinal chemists in their molecular design decisions. Here we demonstrate that protein-bound ligand 1 H NMR chemical shifts are highly sensitive and accurate probes for the immediate chemical environment of protein-ligand interfaces. By comparing the experimental ligand 1 H chemical shift values with those computed from the X-ray structure using quantum mechanics methodology, we identify significant disagreements for parts of the ligand between the two experimental techniques. We show that quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) ensembles can be used to refine initial X-ray co-crystal structures resulting in a better agreement with experimental 1 H ligand chemical shift values. Overall, our findings highlight the usefulness of ligand 1 H NMR chemical shift information in combination with a QM/MM MD workflow for generating protein-ligand ensembles that accurately reproduce solution structural data.

Unraveling the Catalytic Mechanism of ß-Cyclodextrin in the Vitamin D Formation.

Previous experimental studies have shown that the isomerization reaction of previtamin D3 (PreD3) to vitamin D3 (VitD3) is accelerated 40-fold when it takes place within a ß-cyclodextrin dimer, in comparison to the reaction occurring in conventional isotropic solutions. In this study, we employ quantum mechanics-based molecular dynamics (MD) simulations and statistical multistructural variational transition state theory to unveil the origin of this acceleration. We find that the conformational landscape in the PreD3 isomerization is highly dependent on whether the system is encapsulated. In isotropic media, the triene moiety of the PreD3 exhibits a rich torsional flexibility. However, when encapsulated, such a flexibility is limited to a more confined conformational space. In both scenarios, our calculated rate constants are in close agreement with experimental results and allow us to identify the PreD3 flexibility restriction as the primary catalytic factor. These findings enhance our understanding of VitD3 isomerization and underscore the significance of MD and environmental factors in biochemical modeling.

PyrAtes: Modular Organic Salts with Large Stokes Shifts for Fluo-rescence Microscopy.

The deployment of small-molecule fluorescent agents plays an ever-growing role in medicine and drug development. Herein, we complement the portfolio of powerful fluorophores, reporting the serendipitous discovery and development of a novel class with an imidazo[1,2-a]pyridinium triflate core, which we term PyrAtes. These fluorophores are synthesized in a single step from readily available materials (>60 examples) and display Stokes shifts as large as 240 nm, while also reaching NIR-I emissions at λmax as long as 720 nm. Computational studies allow the development of a platform for the prediction of λmax and λEm. Furthermore, we demonstrate the compatibility of these novel fluorophores with live cell imaging in HEK293 cells, suggesting PyrAtes as potent intracellular markers.

Prediction of Enzyme Catalysis by Computing Reaction Energy Barriers via Steered QM/MM Molecular Dynamics Simulations and Machine Learning.

The prediction of enzyme activity is one of the main challenges in catalysis. With computer-aided methods, it is possible to simulate the reaction mechanism at the atomic level. However, these methods are usually expensive if they are to be used on a large scale, as they are needed for protein engineering campaigns. To alleviate this situation, machine learning methods can help in the generation of predictive-decision models. Herein, we test different regression algorithms for the prediction of the reaction energy barrier of the rate-limiting step of the hydrolysis of mono-(2-hydroxyethyl)terephthalic acid by the MHETase ofIdeonella sakaiensis. As a training data set, we use steered quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulation snapshots and their corresponding pulling work values. We have explored three algorithms together with three chemical representations. As an outcome, our trained models are able to predict pulling works along the steered QM/MM MD simulations with a mean absolute error below 3 kcal mol-1 and a score value above 0.90. More challenging is the prediction of the energy maximum with a single geometry. Whereas the use of the initial snapshot of the QM/MM MD trajectory as input geometry yields a very poor prediction of the reaction energy barrier, the use of an intermediate snapshot of the former trajectory brings the score value above 0.40 with a low mean absolute error (ca. 3 kcal mol-1). Altogether, we have faced in this work some initial challenges of the final goal of getting an efficient workflow for the semiautomatic prediction of enzyme-catalyzed energy barriers and catalytic efficiencies.

Unravelling the Turn-On Fluorescence Mechanism of a Fluorescein-Based Probe in GABAA Receptors.

GABAA (γ-aminobutyric acid type A) receptors are ligand-gated ion channels mediating fast inhibitory transmission in the mammalian brain. Here we report the molecular and electronic mechanism governing the turn-on emission of a fluorescein-based imaging probe able to target the human GABAA receptor. Multiscale calculations evidence a drastic conformational change of the probe from folded in solution to extended upon binding to the receptor. Intramolecular ππ-stacking interactions present in the folded probe are responsible for quenching fluorescence in solution. In contrast, unfolding within the GABAA receptor changes the nature of the bright excited state triggering emission. Remarkably, this turn-on effect only manifests for the dianionic prototropic form of the imaging probe, which is found to be the strongest binder to the GABAA receptor. This study is expected to assist the design of new photoactivatable screening tools for allosteric modulators of the GABAA receptor.

Deciphering the Chemical Basis of Fluorescence of a Selenium-Labeled Uracil Probe when Bound at the Bacterial Ribosomal A-Site.

We unveil in this work the main factors that govern the turn-on/off fluorescence of a Se-modified uracil probe at the ribosomal RNA A-site. Whereas the constraint into an "in-plane" conformation of the two rings of the fluorophore is the main driver for the observed turn-on fluorescence emission in the presence of the antibiotic paromomycin, the electrostatics of the environment plays a minor role during the emission process. Our computational strategy clearly indicates that, in the absence of paromomycin, the probe prefers conformations that show a dark S1 electronic state with participation of nπ* electronic transition contributions between the selenium atom and the π-system of the uracil moiety.

Spiropyran Meets Guanine Quadruplexes: Isomerization Mechanism and DNA Binding Modes of Quinolizidine-Substituted Spiropyran Probes.

The recent delivery of a fluorescent quinolizidine-substituted spiropyran, which is able to switch in vivo and bind to guanine quadruplexes (G4) at physiological pH values, urged us to elucidate its molecular switching and binding mechanism. Combining multiscale dynamical studies and accurate quantum chemical calculations, we show that, both in water and in the G4 environment, the switching of the spiropyran ring is not promoted by an initial protonation event-as expected by the effect of low pH solutions-but that the deprotonated merocyanine form is an intermediate of the reaction leading to the protonated open species. Additionally, we investigate the binding of both deprotonated and protonated open forms of merocyanine to c-MYC G4s. Both species bind to G4s albeit with different hydrogen-bond patterns and provide distinct rotamers around the exocyclic double bond of the merocyanine forms. Altogether, our study sheds light on the pharmacophoric points for the binding of these probes to DNA, and thereby, contributes to future developments of new G4 binders of the remarkable family of quinolizidine-substituted spiropyrans.

A Supramolecular Stabilizer of the 14-3-3ζ/ERα Protein-Protein Interaction with a Synergistic Mode of Action.

We report on a stabilizer of the interaction between 14-3-3ζ and the Estrogen Receptor alpha (ERα). ERα is a driver in the majority of breast cancers and 14-3-3 proteins are negative regulators of this nuclear receptor, making the stabilization of this protein-protein interaction (PPI) an interesting strategy. The stabilizer (1) consists of three symmetric peptidic arms containing an arginine mimetic, previously described as the GCP motif. 1 stabilizes the 14-3-3ζ/ERα interaction synergistically with the natural product Fusicoccin-A and was thus hypothesized to bind to a different site. This is supported by computational analysis of 1 binding to the binary complex of 14-3-3 and an ERα-derived phosphopeptide. Furthermore, 1 shows selectivity towards 14-3-3ζ/ERα interaction over other 14-3-3 client-derived phosphomotifs. These data provide a solid support of a new binding mode for a supramolecular 14-3-3ζ/ERα PPI stabilizer.

Reaction mechanism of nucleoside 2'-deoxyribosyltransferases: free-energy landscape supports an oxocarbenium ion as the reaction intermediate.

Insight into the catalytic mechanism of Lactobacillus leichmannii nucleoside 2'-deoxyribosyltransferase (LlNDT) has been gained by calculating a quantum mechanics-molecular mechanics (QM/MM) free-energy landscape of the reaction within the enzyme active site. Our results support an oxocarbenium species as the reaction intermediate and thus an SN1 reaction mechanism in this family of bacterial enzymes. Our mechanistic proposal is validated by comparing experimental kinetic data on the impact of the single amino acid replacements Tyr7, Glu98 and Met125 with Ala, Asp and Ala/norLeu, respectively, and accounts for the specificity shown by this enzyme on a non-natural substrate. This work broadens our understanding of enzymatic C-N bond cleavage and C-N bond formation.

Cold-induced aldimine bond cleavage by Tris in Bacillus subtilis alanine racemase.

Pyridoxal 5'-phosphate (PLP) is a versatile cofactor involved in a large variety of enzymatic processes. Most of PLP-catalysed reactions, such as those of alanine racemases (AlaRs), present a common resting state in which the PLP is covalently bound to an active-site lysine to form an internal aldimine. The crystal structure of BsAlaR grown in the presence of Tris lacks this covalent linkage and the PLP cofactor appears deformylated. However, loss of activity in a Tris buffer only occurred after the solution was frozen prior to carrying out the enzymatic assay. This evidence strongly suggests that Tris can access the active site at subzero temperatures and behave as an alternate racemase substrate leading to mechanism-based enzyme inactivation, a hypothesis that is supported by additional X-ray structures and theoretical results from QM/MM calculations. Taken together, our findings highlight a possibly underappreciated role for a common buffer component widely used in biochemical and biophysical experiments.

Arginine mimetic appended peptide-based probes for fluorescence turn-on detection of 14-3-3 proteins.

14-3-3 proteins are adaptor elements in intracellular signaling pathways. Recently, this protein family has been identified as a relevant therapeutic target involved in many human diseases. Therefore, identification of 14-3-3 proteins in biological systems is very important. Two cationic peptide-based probes are reported for the fluorescence detection of 14-3-3 proteins at physiological pH. The design of these probes consists of two symmetric peptidic arms equipped with a guanidiniocarbonyl pyrrole moiety (an arginine mimetic aka GCP), and an environment-sensitive amino-naphthalimide fluorophore as a third arm. These peptide sequences also contain lysine and phenylalanine/tryptophan amino acids for additional charge-charge and hydrophobic interactions. Both probes show high affinity and sensitivity for the 14-3-3 family, as well as good selectivity against other relevant biological proteins and ions.

Unravelling the covalent binding of zampanolide and taccalonolide AJ to a minimalist representation of a human microtubule.

Many natural products target mammalian tubulin but only a few can form a covalent bond and hence irreversibly affect microtubule function. Among them, zampanolide (ZMP) and taccalonolide AJ (TAJ) stand out, not only because they are very potent antitumor agents but also because the adducts they form with ß-tubulin have been structurally characterized in atomic detail. By applying model building techniques, molecular orbital calculations, molecular dynamics simulations and hybrid QM/MM methods, we have gained insight into the 1,2- and 1,4-addition reactions of His229 and Asp226 to ZMP and TAJ, respectively, in the taxane-binding site of ß-tubulin. The experimentally inaccessible precovalent complexes strongly suggest a water-mediated proton shuttle mechanism for ZMP adduct formation and a direct nucleophilic attack by the carboxylate of Asp226 on C22 of the C22R,C23R epoxide in TAJ. The M-loop, which is crucially important for interprotofilament interactions, is structured into a short helix in both types of complexes, mostly as a consequence of the fixation of the phenol ring of Tyr283 and the guanidinium of Arg284. As a side benefit, we obtained evidence supporting the existence of a commonly neglected intramolecular disulfide bond between Cys241 and Cys356 in ß-tubulin that contributes to protein compactness and is absent in the ßIII isotype associated with resistance to taxanes and other drugs.

Directional and regioselective hole injection of spiropyran photoswitches intercalated into A/T-duplex DNA.

The electron-hole injection from a family of spiropyran photoswitches into A/T-duplex DNA has been investigated at the molecular level for the first time. Multiscale computations coupled with automatized quantitative wavefunction analysis reveal a pronounced directionality and regioselectivity towards the template strand of the duplex DNA. Our findings suggest that this directional and regioselective photoinduced electron-hole transfer could thus be exploited to tailor the charge transport processes in DNA in specific applications.

DNA-binding mechanism of spiropyran photoswitches: the role of electrostatics.

The binding mechanism of the protonated open form of three spiropyran derivatives into a 12-mer (poly-dAT)2 has been unveiled by means of computational methods. It is found that the electrostatic term in the probe:DNA binding energy, modulates the binding mode, providing new guidelines for the design of spiropyran photoswitches with specific binding modes to DNA.

Structure-based domain assignment in Leishmania infantum EndoG: characterization of a pH-dependent regulatory switch and a C-terminal extension that largely dictates DNA substrate preferences.

Mitochondrial endonuclease G from Leishmania infantum (LiEndoG) participates in the degradation of double-stranded DNA (dsDNA) during parasite cell death and is catalytically inactive at a pH of 8.0 or above. The presence, in the primary sequence, of an acidic amino acid-rich insertion exclusive to trypanosomatids and its spatial position in a homology-built model of LiEndoG led us to postulate that this peptide stretch might act as a pH sensor for self-inhibition. We found that a LiEndoG variant lacking residues 145-180 is indeed far more active than its wild-type counterpart at pH values >7.0. In addition, we discovered that (i) LiEndoG exists as a homodimer, (ii) replacement of Ser211 in the active-site SRGH motif with the canonical aspartate from the DRGH motif of other nucleases leads to a catalytically deficient enzyme, (iii) the activity of the S211D variant can be restored upon the concomitant replacement of Ala247 with Arg and (iv) a C-terminal extension is responsible for the observed preferential cleavage of single-stranded DNA (ssDNA) and ssDNA-dsDNA junctions. Taken together, our results support the view that LiEndoG is a multidomain molecular machine whose nuclease activity can be subtly modulated or even abrogated through architectural changes brought about by environmental conditions and interaction with other binding partners.

Engineering Erg10 Thiolase from Saccharomyces cerevisiae as a Synthetic Toolkit for the Production of Branched-Chain Alcohols.

Thiolases catalyze the condensation of acyl-CoA thioesters through the Claisen condensation reaction. The best described enzymes usually yield linear condensation products. Using a combined computational/experimental approach, and guided by structural information, we have studied the potential of thiolases to synthesize branched compounds. We have identified a bulky residue located at the active site that blocks proper accommodation of substrates longer than acetyl-CoA. Amino acid replacements at such a position exert effects on the activity and product selectivity of the enzymes that are highly dependent on a protein scaffold. Among the set of five thiolases studied, Erg10 thiolase from Saccharomyces cerevisiae showed no acetyl-CoA/butyryl-CoA branched condensation activity, but variants at position F293 resulted the most active and selective biocatalysts for this reaction. This is the first time that a thiolase has been engineered to synthesize branched compounds. These novel enzymes enrich the toolbox of combinatorial (bio)chemistry, paving the way for manufacturing a variety of α-substituted synthons. As a proof of concept, we have engineered Clostridium's 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol that is interesting as a branched model compound.

A Series of Enthalpically Optimized Docetaxel Analogues Exhibiting Enhanced Antitumor Activity and Water Solubility.

A dual-purpose strategy aimed at enhancing the binding affinity for microtubules and improving the water solubility of docetaxel led to the design and synthesis of a series of C-2- and C-3'-modified analogues. Both aims were realized when the C-3' phenyl group present in docetaxel was replaced with a propargyl alcohol. The resulting compound, 3f, was able to overcome drug resistance in cultured P-gp-overexpressing tumor cells and showed greater activity than docetaxel against drug-resistant A2780/AD ovarian cancer xenografts in mice. In addition, the considerably lower hydrophobicity of 3f relative to both docetaxel and paclitaxel led to better aqueous solubility. A molecular model of tubulin-bound 3f revealed novel hydrogen-bonding interactions between the propargyl alcohol and the polar environment provided by the side chains of Ser236, Glu27, and Arg320.

Structural rationale for the cross-resistance of tumor cells bearing the A399V variant of elongation factor eEF1A1 to the structurally unrelated didemnin B, ternatin, nannocystin A and ansatrienin B.

At least four classes of structurally distinct natural products with potent antiproliferative activities target the translation elongation factor eEF1A1, which is best known as the G-protein that delivers amino acyl transfer RNAs (aa-tRNAs) to ribosomes during mRNA translation. We present molecular models in atomic detail that provide a common structural basis for the high-affinity binding of didemnin B, ternatin, ansatrienin B and nannocystin A to eEF1A1, as well as a rationale based on molecular dynamics results that accounts for the deleterious effect of replacing alanine 399 with valine. The proposed binding site, at the interface between domains I and III, is eminently hydrophobic and exists only in the GTP-bound conformation. Drug binding at this site is expected to disrupt neither loading of aa-tRNAs nor GTP hydrolysis but would give rise to stabilization of this particular conformational state, in consonance with reported experimental findings. The experimental solution of the three-dimensional structure of mammalian eEF1A1 has proved elusive so far and the highly homologous eEF1A2 from rabbit muscle has been crystallized and solved only as a homodimer in a GDP-bound conformation. Interestingly, in this dimeric structure the large interdomain cavity where the drugs studied are proposed to bind is occupied by a mostly hydrophobic α-helix from domain I of the same monomer. Since binding of this α-helix and any of these drugs to domain III of eEF1A(1/2) is, therefore, mutually exclusive and involves two distinct protein conformations, one intriguing possibility that emerges from our study is that the potent antiproliferative effect of these natural products may arise not only from inhibition of protein synthesis, which is the current dogma, but also from interference with some other non-canonical functions. From this standpoint, this type of drugs could be considered antagonists of eEF1A1/2 oligomerization, a hypothesis that opens up novel areas of research.

Making sense of the past: hyperstability of ancestral thioredoxins explained by free energy simulations.

Thioredoxin (Trx), a small and globular protein, is present in all kinds of organisms, from Archea to higher mammals. Throughout evolution, the Trx sequence has undergone subtle modifications to adapt to varying environmental conditions. The high degree of sequence conservation makes Trx very amenable to ancestral protein reconstruction techniques. In this work, we address the study of the structural and energetic determinants of thermostability in E. coli Trx using a dataset of mutations inspired by ancestral reconstruction. We compute, from first principles, the expected contribution of 19 different amino acid substitutions to the stability (ΔΔG) and the melting temperature (ΔTm) of the protein. We also describe the specific changes in structure and protein dynamics responsible for the stabilizing or destabilizing effects of these mutations. Our results point to local and independent changes for most of the variants. Our predictions are accurate enough to substantiate the proposal of new hypotheses regarding evolutionary relationships between mutations, as in the case of T89R, P68A and G74S or K90L and F102A, and reach beyond the initial set to suggest improved variants, such as K90I or K90Y.

Stepwise Simulation of 3,5-Dihydro-5-methylidene-4H-imidazol-4-one (MIO) Biogenesis in Histidine Ammonia-lyase.

A 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) electrophilic moiety is post-translationally and autocatalytically generated in homotetrameric histidine ammonia-lyase (HAL) and other enzymes containing the tripeptide Ala-Ser-Gly in a suitably positioned loop. The backbone cyclization step is identical to that taking place during fluorophore formation in green fluorescent protein from the tripeptide Ser-Tyr-Gly, but dehydration, rather than dehydrogenation by molecular oxygen, is the reaction that gives rise to the mature MIO ring system. To gain additional insight into this unique process and shed light on some still unresolved issues, we have made use of extensive molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations implementing the self-consistent charge density functional tight-binding method on a fully solvated tetramer of Pseudomonas putida HAL. Our results strongly support the idea that mechanical compression of the reacting loop by neighboring protein residues in the precursor state is absolutely required to prevent formation of inhibitory main-chain hydrogen bonds and to enforce proper alignment of donor and acceptor orbitals for bond creation. The consideration of the protein environment in our computations shows that water molecules, which have been mostly neglected in previous theoretical work, play a highly relevant role in the reaction mechanism and, more importantly, that backbone cyclization resulting from the nucleophilic attack of the Gly amide lone pair on the π* orbital of the Ala carbonyl precedes side-chain dehydration of the central serine.