Human indoleamine-2,3-dioxygenase 2 cofactor lability and low substrate affinity explained by homology modeling, molecular dynamics and molecular docking.

The human indoleamine-2,3-dioxygenase 2 (hIDO2) protein is growing of interest as it is increasingly implicated in multiple diseases (cancer, autoimmune diseases, COVID-19). However, it is only poorly reported in the literature. Its mode of action remains unknown because it does not seem to catalyze the reaction for which it is attributed: the degradation of the L-Tryptophan into N-formyl-kynurenine. This contrasts with its paralog, the human indoleamine-2,3-dioxygenase 1 (hIDO1), which has been extensively studied in the literature and for which several inhibitors are already in clinical trials. Yet, the recent failure of one of the most advanced hIDO1 inhibitors, the Epacadostat, could be caused by a still unknown interaction between hIDO1 and hIDO2. In order to better understand the mechanism of hIDO2, and in the absence of experimental structural data, a computational study mixing homology modeling, Molecular Dynamics, and molecular docking was conducted. The present article highlights an exacerbated lability of the cofactor as well as an inadequate positioning of the substrate in the active site of hIDO2, which might bring part of an answer to its lack of activity.Communicated by Ramaswamy H. Sarma.

Description of non-covalent interactions in benzyl chalcocyanate crystals from smoothed Cromer-Mann electron density distribution functions.

A well-known method to characterize non-covalent interactions consists in the topological analysis of electron density distribution (EDD) functions, complemented by the search for minima in the reduced density gradient (RDG) distributions. Here, we characterize intermolecular interactions occurring in crystals of benzyl chalcocyanate compounds through bond critical points (BCP) of the promolecular electron density (ED) built from the crystallographic Cromer-Mann parameters, at several smoothing levelst. The trajectories formed by thet-dependent BCP locations are interpreted in terms of the intermolecular interactions occurring within the crystal arrangements. Chalcogen…nitro BCPs are clearly present in the unsmoothed EDDs but are annihilated astincreases, while chalcogen…chalcogen BCPs appear and are among the only BCPs left at the highest smoothing level. The chalcogen bonds are differentiated from the other chalcogen interactions through the linear chalcogen…BCP…nitro geometry at low smoothing level and their more negative Laplacian values. The annihilation of CPs can be followed by the apparition of a RDG minimum, associated with a very weak interaction. Along the BCP trajectories, the Laplacian shows a progressive concentration of the ED in the intermolecular space within the crystals and adopts the most negative values at the shortest atom…atom separations. At the termination point of a BCP trajectory, the drastic increase of the ellipticity value illustrates the flattening of the EDD.

Temporary Intermediates of L-Trp Along the Reaction Pathway of Human Indoleamine 2,3-Dioxygenase 1 and Identification of an Exo Site.

Protein dynamics governs most of the fundamental processes in the human body. Particularly, the dynamics of loops located near an active site can be involved in the positioning of the substrate and the reaction mechanism. The understanding of the functioning of dynamic loops is therefore a challenge, and often requires the use of a multi-disciplinary approach mixing, for example, crystallographic experiments and molecular dynamics simulations. In the present work, the dynamic behavior of the JK-loop of the human indoleamine 2,3-dioxygenase 1 hemoprotein, a target for immunotherapy, is investigated. To overcome the lack of knowledge on this dynamism, the study reported here is based on 3 crystal structures presenting different conformations of the loop, completed with molecular dynamics trajectories and MM-GBSA analyses, in order to trace the reaction pathway of the enzyme. In addition, the crystal structures identify an exo site in the small unit of the enzyme, that is populated redundantly by the substrate or the product of the reaction. The role of this newer reported exo site still needs to be investigated.

Structure and Dynamics of an Archeal Monoglyceride Lipase from Palaeococcus ferrophilus as Revealed by Crystallography and In Silico Analysis.

The crystallographic analysis of a lipase from Palaeococcus ferrophilus (PFL) previously annotated as a lysophospholipase revealed high structural conservation with other monoglyceride lipases, in particular in the lid domain and substrate binding pockets. In agreement with this observation, PFL was shown to be active on various monoacylglycerols. Molecular Dynamics (MD) studies performed in the absence and in the presence of ligands further allowed characterization of the dynamics of this system and led to a systematic closure of the lid compared to the crystal structure. However, the presence of ligands in the acyl-binding pocket stabilizes intermediate conformations compared to the crystal and totally closed structures. Several lid-stabilizing or closure elements were highlighted, i.e., hydrogen bonds between Ser117 and Ile204 or Asn142 and its facing amino acid lid residues, as well as Phe123. Thus, based on this complementary crystallographic and MD approach, we suggest that the crystal structure reported herein represents an open conformation, at least partially, of the PFL, which is likely stabilized by the ligand, and it brings to light several key structural features prone to participate in the closure of the lid.

Structure-based identification of inhibitors disrupting the CD2-CD58 interactions.

The immune system has very intricate mechanisms of fighting against the invading infections which are accomplished by a sequential event of molecular interactions in the body. One of the crucial phenomena in this process is the recognition of T-cells by the antigen-presenting cells (APCs), which is initiated by the rapid interaction between both cell surface receptors, i.e., CD2 located on T-cells and CD58 located on APCs. Under various pathological conditions, which involve undesired immune response, inhibiting the CD2-CD58 interactions becomes a therapeutically relevant opportunity. Herein we present an extensive work to identify novel inhibiting agents of the CD2-CD58 interactions. Classical molecular dynamics (MD) simulations of the CD2-CD58 complex highlighted a series of crucial CD58 residues responsible for the interactions with CD2. Based on such results, a pharmacophore map, complementary to the CD2-binding site of CD58, was created and employed for virtual screening of ~ 300,000 available compounds. On the ~ 6000 compounds filtered from pharmacophore mapping, ADME screening leads to ~ 350 molecules. Molecular docking was then performed on these molecules, and fifteen compounds emerged with significant binding energy (< - 50 kcal/mol) for CD58. Finally, short MD simulations were performed in triplicate on each complex (i) to provide a microscopic view of the ligand binding and (ii) to rule out possibly weak binders of CD58 from the identified hits. At last, we suggest eight compounds for in vitro testing that were identified as promising hits to bind CD58 with a high binding affinity.

Molecular Mechanism for the Self-Supported Synthesis of Graphitic Carbon Nitride from Urea Pyrolysis.

Quantum chemical calculations combined with kinetic Monte Carlo simulations are performed to decipher the kinetics for the one-pot synthesis of two-dimensional graphitic carbon nitride (g-C3N4) from urea pyrolysis. Two mechanisms are considered, one involving ammelide as the intermediate compound and the other considering cyanuric acid. Different grid growing patterns are investigated, and the size, shape, and density of the grids as well as the number and position of the defects are evaluated. We find that the mechanistic pathway involving ammelide is preferred. Larger g-C3N4 grids with lower density are achieved when the rate constant for melon growing is inversely proportional to the number of local reaction sites, while nearly filled smaller grids are obtained in the opposite scenario. Larger defects appear at the grid periphery while smaller holes appear throughout the grid. The synthesis of extended g-C3N4 structures is favored if the g-C3N4 growing propensity is directly proportional to the number of reaction sites.

Investigation of bound and unbound phosphoserine phosphatase conformations through elastic network models and molecular dynamics simulations.

The human phosphoserine phosphatase (hPSP) catalyses the last step in the biosynthesis of L-serine. It involves conformational changes of the enzyme lid once the substrate, phosphoserine (PSer), is bound in the active site. Here, Elastic Network Model (ENM) is applied to the crystal structure of hPSP to probe the transition between open and closed conformations of hPSP. Molecular Dynamics (MD) simulations are carried out on several PSer-hPSP systems to characterise the intermolecular interactions and their effect on the dynamics of the enzyme lid. Systems involving either Ca++ or Mg++ are considered. The first ENM normal mode shows that an open-closed transition can be explained from a simple description of the enzyme in terms of harmonic potentials. Principal Component Analyses applied to the MD trajectories also highlight a trend for a closing/opening motion. Different PSer orientations inside the enzyme cavity are identified, i.e. either the carboxylate, the phosphate group of PSer, or both, are oriented towards the cation. The interaction patterns are analysed in terms of hydrogen bonds, electrostatics, and bond critical points of the electron density distributions. The latter approach yields a global description of the bonding intermolecular interactions. The PSer orientation determines the content of the cation coordination shell and the mobility of the substrate, while Lys158 and Thr182, involved in the reaction mechanism, are always in interaction with the substrate. Closed enzyme conformations involve Met52-Gln204, Arg49-Glu29, and Arg50-Glu29 interactions. Met52, as well as Arg49 and Arg50, also stabilize PSer inside the cavity. Communicated by Ramaswamy H. Sarma.

Influence of the presence of the heme cofactor on the JK-loop structure in indoleamine 2,3-dioxygenase 1.

Indoleamine 2,3-dioxygenase 1 has sparked interest as an immunotherapeutic target in cancer research. Its structure includes a loop, named the JK-loop, that controls the orientation of the substrate or inhibitor within the active site. However, little has been reported about the crystal structure of this loop. In the present work, the conformation of the JK-loop is determined for the first time in the presence of the heme cofactor in the active site through X-ray diffraction experiments (2.44 Šresolution). Molecular-dynamics trajectories were also obtained to provide dynamic information about the loop according to the presence of cofactor. This new structural and dynamic information highlights the importance of the JK-loop in confining the labile heme cofactor to the active site.

Crystal structures and snapshots along the reaction pathway of human phosphoserine phosphatase.

The equilibrium between phosphorylation and dephosphorylation is one of the most important processes that takes place in living cells. Human phosphoserine phosphatase (hPSP) is a key enzyme in the production of serine by the dephosphorylation of phospho-L-serine. It is directly involved in the biosynthesis of other important metabolites such as glycine and D-serine (a neuromodulator). hPSP is involved in the survival mechanism of cancer cells and has recently been found to be an essential biomarker. Here, three new high-resolution crystal structures of hPSP (1.5-2.0 Å) in complexes with phosphoserine and with serine, which are the substrate and the product of the reaction, respectively, and in complex with a noncleavable substrate analogue (homocysteic acid) are presented. New types of interactions take place between the enzyme and its ligands. Moreover, the loop involved in the open/closed state of the enzyme is fully refined in a totally unfolded conformation. This loop is further studied through molecular-dynamics simulations. Finally, all of these analyses allow a more complete reaction mechanism for this enzyme to be proposed which is consistent with previous publications on the subject.

Interaction of POPC, DPPC, and POPE with the µ opioid receptor: A coarse-grained molecular dynamics study.

The µ opioid receptor (µOR), which is part of the G protein-coupled receptors family, is a membrane protein that is modulated by its lipid environment. In the present work, we model µOR in three different membrane systems: POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), and DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphocholine) through 45 µs molecular dynamics (MD) simulations at the coarse-grained level. Our theoretical studies provide new insights to the lipid-induced modulation of the receptor. Particularly, to characterize how µOR interacts with each lipid, we analyze the tilt of the protein, the number of contacts occurring between the lipids and each amino acid of the receptor, and the µOR-lipid interface described as a network graph. We also analyze the variations in the number and the nature of the protein contacts that are induced by the lipid structure. We show that POPC interacts preferentially with helix 1 (H1) and helices H5-H6, POPE, with H5-H6 and H6-H7, and DPPC, with H4 and H6. We demonstrate how each of the three lipids shape the structure of the µOR.

Investigating cyclic peptides inhibiting CD2-CD58 interactions through molecular dynamics and molecular docking methods.

The CD2-CD58 protein-protein interaction is known to favor the recognition of antigen presenting cells by T cells. The structural, energetics, and dynamical properties of three known cyclic CD58 ligands, named P6, P7, and RTD-c, are studied through molecular dynamics (MD) simulations and molecular docking calculations. The ligands are built so as to mimic the C and F ß-strands of protein CD2, connected via turn inducers. The MD analyses focus on the location of the ligands with respect to the experimental binding site and on the direct and water-mediated hydrogen bonds (H bonds) they form with CD58. Ligand P6, with a sequence close to the experimental ß-strands of CD2, presents characteristics that explain its higher experimental affinity, e.g., the lower mobility and flexibility at the CD58 surface, and the larger number and occurrence frequency of ligand-CD58 H bonds. For the two other ligands, the structural modifications lead to changes in the binding pattern with CD58 and its dynamics. In parallel, a large set of molecular docking calculations, carried out with various search spaces and docking algorithms, are compared to provide a consensus view of the preferred ligand binding modes. The analysis of the ligand side chain locations yields results that are consistent with the CD2-CD58 crystal structure and suggests various binding modes of the experimentally identified hot spot of the ligands, i.e., Tyr86. P6 is shown to form a number of contacts that are also present in the experimental CD2-CD58 structure.

Reduced Point Charge Models of Proteins: Effect of Protein-Water Interactions in Molecular Dynamics Simulations of Ubiquitin Systems.

We investigate the influence of various solvent models on the structural stability and protein-water interface of three ubiquitin complexes (PDB access codes: 1Q0W , 2MBB , 2G3Q ) modeled using the Amber99sb force field (FF) and two different point charge distributions. A previously developed reduced point charge model (RPCM), wherein each amino acid residue is described by a limited number of point charges, is tested and compared to its all-atom (AA) version. The complexes are solvated in TIP4P-Ew or TIP3P type water molecules, involving either the scaling of the Lennard-Jones protein-Owater interaction parameters, or the coarse-grain (CG) SIRAH water description. The best agreements between the RPCM and AA models were obtained for structural, protein-water, and ligand-ubiquitin properties when using the TIP4P-Ew water FF with a scaling factor γ of 0.7. At the RPCM level, a decrease in γ, or the inclusion of SIRAH particles, allows weakening of the protein-water interactions. It results in a slight collapse of the protein structure and a less compact hydration shell and, thus, in a decrease in the number of protein-water and water-water H-bonds. The dynamics of the surface protein atoms and of the water shell molecules are also slightly refrained, which allow the generation of stable RPCM trajectories.

Parameterization of the ReaxFF reactive force field for a proline-catalyzed aldol reaction.

A parameterization of the ReaxFF reactive FF is performed using a Monte Carlo Simulated Annealing procedure for the modeling of a proline-catalyzed aldol reaction. Emphasis is put on the accurate reproduction of the relative stabilities of several key intermediates of the reaction, as well as, on the description of the reaction path bridging these intermediates based on quantum mechanical calculations. Our training sets include new criteria based on geometry optimizations and short Molecular Dynamics simulations to ensure that the trained ReaxFF potentials adequately predict the structures of all key intermediates. The transferability of the sets of parameters obtained is assessed for various steps of the considered aldol reaction, as well as for different substrates, catalysts, and reagents. This works indeed highlights the challenge of reaching transferable parameters for several reaction steps. © 2016 Wiley Periodicals, Inc.

Multiscale design of coarse-grained elastic network-based potentials for the µ opioid receptor.

Despite progress in computer modeling, most biological processes are still out of reach when using all-atom (AA) models. Coarse-grained (CG) models allow classical molecular dynamics (MD) simulations to be accelerated. Although simplification of spatial resolution at different levels is often investigated, simplification of the CG potential in itself has been less common. CG potentials are often similar to AA potentials. In this work, we consider the design and reliability of purely mechanical CG models of the µ opioid receptor (µOR), a G protein-coupled receptor (GPCR). In this sense, CG force fields (FF) consist of a set of holonomic constraints guided by an elastic network model (ENM). Even though ENMs are used widely to perform normal mode analysis (NMA), they are not often implemented as a single FF in the context of MD simulations. In this work, various ENM-like potentials were investigated by varying their force constant schemes and connectivity patterns. A method was established to systematically parameterize ENM-like potentials at different spatial resolutions by using AA data. To do so, new descriptors were introduced. The choice of conformation descriptors that also include flexibility information is important for a reliable parameterization of ENMs with different degrees of sensitivity. Hence, ENM-like potentials, with specific parameters, can be sufficient to accurately reproduce AA MD simulations of µOR at highly coarse-grained resolutions. Therefore, the essence of the flexibility properties of µOR can be captured with simple models at different CG spatial resolutions, opening the way to mechanical approaches to understanding GPCR functions. Graphical Abstract All atom structure, residue interaction network and coarse-grained elastic network models of the µ opioid receptor (µOR).

Modeling of structural, energetic, and dynamic properties of few-atom silver clusters embedded in polynucleotide strands by using molecular dynamics.

This work concerns the study of the structural, energetic, and dynamic properties of fluorescent systems composed of silver clusters stabilized by polynucleotide strands. To do so, classical interaction potentials relative to silver, neutral and cationic, were introduced in the AMBER force field. Molecular dynamics simulations allowed analysis of the nature and force of the interactions between the various parts of the nucleic oligomers and the silver clusters. Conformational analyses were necessary to explore the flexibility of the supramolecular assemblies, specifically by radial distribution functions and Ramachandran-type maps.

On the modularity of the intrinsic flexibility of the µ opioid receptor: a computational study.

The µ opioid receptor (µOR), the principal target to control pain, belongs to the G protein-coupled receptors (GPCRs) family, one of the most highlighted protein families due to their importance as therapeutic targets. The conformational flexibility of GPCRs is one of their essential characteristics as they take part in ligand recognition and subsequent activation or inactivation mechanisms. It is assessed that the intrinsic mechanical properties of the µOR, more specifically its particular flexibility behavior, would facilitate the accomplishment of specific biological functions, at least in their first steps, even in the absence of a ligand or any chemical species usually present in its biological environment. The study of the mechanical properties of the µOR would thus bring some indications regarding the highly efficient ability of the µOR to transduce cellular message. We therefore investigate the intrinsic flexibility of the µOR in its apo-form using all-atom Molecular Dynamics simulations at the sub-microsecond time scale. We particularly consider the µOR embedded in a simplified membrane model without specific ions, particular lipids, such as cholesterol moieties, or any other chemical species that could affect the flexibility of the µOR. Our analyses highlighted an important local effect due to the various bendability of the helices resulting in a diversity of shape and volume sizes adopted by the µOR binding site. Such property explains why the µOR can interact with ligands presenting highly diverse structural geometry. By investigating the topology of the µOR binding site, a conformational global effect is depicted: the correlation between the motional modes of the extra- and intracellular parts of µOR on one hand, along with a clear rigidity of the central µOR domain on the other hand. Our results show how the modularity of the µOR flexibility is related to its pre-ability to activate and to present a basal activity.

Evaluation of reduced point charge models of proteins through Molecular Dynamics simulations: application to the Vps27 UIM-1-Ubiquitin complex.

Reduced point charge models of amino acids are designed, (i) from local extrema positions in charge density distribution functions built from the Poisson equation applied to smoothed molecular electrostatic potential (MEP) functions, and (ii) from local maxima positions in promolecular electron density distribution functions. Corresponding charge values are fitted versus all-atom Amber99 MEPs. To easily generate reduced point charge models for protein structures, libraries of amino acid templates are built. The program GROMACS is used to generate stable Molecular Dynamics trajectories of an Ubiquitin-ligand complex (PDB: 1Q0W), under various implementation schemes, solvation, and temperature conditions. Point charges that are not located on atoms are considered as virtual sites with a nul mass and radius. The results illustrate how the intra- and inter-molecular H-bond interactions are affected by the degree of reduction of the point charge models and give directions for their implementation; a special attention to the atoms selected to locate the virtual sites and to the Coulomb-14 interactions is needed. Results obtained at various temperatures suggest that the use of reduced point charge models allows to probe local potential hyper-surface minima that are similar to the all-atom ones, but are characterized by lower energy barriers. It enables to generate various conformations of the protein complex more rapidly than the all-atom point charge representation.

Charge density distributions derived from smoothed electrostatic potential functions: design of protein reduced point charge models.

To generate reduced point charge models of proteins, we developed an original approach to hierarchically locate extrema in charge density distribution functions built from the Poisson equation applied to smoothed molecular electrostatic potential (MEP) functions. A charge fitting program was used to assign charge values to the so-obtained reduced representations. In continuation to a previous work, the Amber99 force field was selected. To easily generate reduced point charge models for protein structures, a library of amino acid templates was designed. Applications to four small peptides, a set of 53 protein structures, and four KcsA ion channel models, are presented. Electrostatic potential and solvation free energy values generated by the reduced models are compared with the corresponding values obtained using the original set of atomic charges. Results are in closer agreement with the original all-atom electrostatic properties than those obtained with a previous reduced model that was directly built from the smoothed MEP functions [Leherte and Vercauteren in J Chem Theory Comput 5:3279-3298, 2009].

Implementation of a protein reduced point charge model toward molecular dynamics applications.

A reduced point charge model was developed in a previous work from the study of extrema in smoothed charge density distribution functions generated from the Amber99 molecular electrostatic potential. In the present work, such a point charge distribution is coupled with the Amber99 force field and implemented in the program TINKER to allow molecular dynamics (MD) simulations of proteins. First applications to two polypeptides that involve α-helix and ß-sheet motifs are analyzed and compared to all-atom MD simulations. Two types of coarse-grained (CG)-based trajectories are generated using, on one hand, harmonic bond stretching terms and, on the other hand, distance restraints. Results show that the use of the unrestrained CG conditions are sufficient to preserve most of the secondary structure characteristics but restraints lead to a better agreement between CG and all-atom simulation results such as rmsd, dipole moment, and time-dependent mean square deviation functions.

Coarse Point Charge Models For Proteins From Smoothed Molecular Electrostatic Potentials.

To generate coarse electrostatic models of proteins, we developed an original approach to hierarchically locate maxima and minima in smoothed molecular electrostatic potentials. A charge-fitting program was used to assign charges to the so-obtained reduced representations. Templates are defined to easily generate coarse point charge models for protein structures, in the particular cases of the Amber99 and Gromos43A1 force fields. Applications to four small peptides and to the ion channel KcsA are presented. Electrostatic potential values generated by the reduced models are compared with the corresponding values obtained using the original sets of atomic charges.