Characterization of human nicotinate phosphoribosyltransferase: Kinetic studies, structure prediction and functional analysis by site-directed mutagenesis

Nicotinate phosphoribosyltransferase (NaPRT, EC 2.4.2.11) catalyzes the conversion of nicotinate (Na) to nicotinate mononucleotide, the first reaction of the Preiss-Handler pathway for the biosynthesis of NAD(+). Even though NaPRT activity has been described to be responsible for the ability of Na to increase NAD(+) levels in human cells more effectively than nicotinamide (Nam), so far a limited number of studies on the human NaPRT have appeared. Here, extensive characterization of a recombinant human NaPRT is reported. We determined its major kinetic parameters and assayed the influence of different compounds on its enzymatic activity. In particular, ATP showed an apparent dual stimulation/inhibition effect at low/high substrates saturation, respectively, consistent with a negative cooperativity model, whereas inorganic phosphate was found to act as an activator. Among other metabolites assayed, including nucleotides, nucleosides, and intermediates of carbohydrates metabolism, some showed inhibitory properties, i.e. CoA, several acyl-CoAs, glyceraldehyde 3-phosphate, phosphoenolpyruvate, and fructose 1,6-bisphosphate, whereas dihydroxyacetone phosphate and pyruvate exerted a stimulatory effect. Furthermore, in light of the absence of crystallographic data, we performed homology modeling to predict the protein three-dimensional structure, and molecular docking simulations to identify residues involved in the recognition and stabilization of several ligands. Most of these residues resulted universally conserved among NaPRTs, and, in this study, their importance for enzyme activity was validated through site-directed mutagenesis.     

Molecular modeling of the three-dimensional structure of GLP-1R and its interactions with several agonists

Glucagon-like peptide-1 receptor (GLP-1R) is a promising molecular target for developing drugs treating type 2 diabetes. We have predicted the complete three-dimensional structure of GLP-1R and the binding modes of several GLP-1R agonists, including GLP-1, Boc5, and Cpd1, through a combination of homology modeling, molecular docking, and long-time molecular dynamics simulation on a lipid bilayer. Our model can reasonably interpret the results of a number of mutation experiments regarding GLP-1R as well as the successful modification to GLP-1 by Liraglutide. Our model is also validated by a recently revealed crystal structure of the extracellular domain of GLP-1R. An activation mechanism of GLP-1R agonists is proposed based on the principal component analysis and normal mode analysis on our predicted GLP-1R structure. Before the complete structure of GLP-1R is determined through experimental means, our model may serve as a valuable reference for characterizing the interactions between GLP-1R and its agonists. Figure Comparison of our predicted model of rGLP-1R (left) with the recently revealed crystal structure of hGLP-1R (right)     

Differences in Binding Sites of Two Melatonin Receptors Help to Explain Their Selectivity to Some Melatonin Analogs: A Molecular Modeling Study

Abstract

Numerous diseases have been linked to the malfunction of G-protein coupled receptors (GP-CRs). Their adequate treatment requires rational design of new high-affinity and high-selectivity drugs targeting these receptors. In this work, we report three-dimensional models of the human MT₁ and MT₂ melatonin receptors, members of the GPCR family. The models are based on the X-ray structure of bovine rhodopsin. The computational approach employs an original procedure for optimization of receptor-ligand structures. It includes rotation of one of the transmembrane α-helices around its axis with simultaneous assessment of quality of the resulting complexes according to a number of criteria we have developed for this purpose. The optimal geometry of the receptor-ligand binding is selected based on the analysis of complementarity of hydrophobic/hydrophilic properties between the ligand and its protein environment in the binding site. The elaborated “optimized” models are employed to explore the details of protein-ligand interactions for melatonin and a number of its analogs with known affinity to MT₁ and MT₂ receptors. The models permit rationalization of experimental data, including those that were not used in model building. The perspectives opened by the constructed models and by the optimization procedure in the design of new drugs are discussed.     

Molecular modeling studies on CNG channel from bovine retinal rod: a structural model of the cyclic nucleotide-binding domain

A dimeric model of the cyclic nucleotide-binding domain of the all-alpha homomeric cyclic nucleotide-gated channel from bovine retinal rod is constructed. The model, based on the structure of the fairly homologous catabolite gene activator protein (Weber and Steitz, J Mol Biol 1987;198:311-326), is obtained by use of comparative modeling and molecular dynamics simulations. Our model provides a structural basis for the experimentally measured difference in activity between cAMP and cGMP, as well as the different solvent accessibilities of GLY597 in the complex with cGMP, with cAMP and in the protein in free state. In addition, it provides support for the rearrangement of the domain C helix on ligand binding and releasing proposed by Matulef et al. (Neuron 1999;24:443-452).     

Repurposing FDA-approved drugs as FXR agonists: a structure based in silico pharmacological study

Farnesoid X receptor (FXR) modulates the expression of genes involved in lipid and carbohydrate homeostasis and inflammatory processes. This nuclear receptor is likely a tumor suppressor in several cancers, but its molecular mechanism of suppression is still under study. Several studies reported that FXR agonism increases the survival of colorectal, biliary tract, and liver cancer patients. In addition, FXR expression was shown to be down-regulated in many diseases such as obesity, irritable bowel syndrome, glomerular inflammation, diabetes, proteinuria, and ulcerative colitis. Therefore, development of novel FXR agonists may have significant potential in the prevention and treatment of these diseases. In this scenario, computer-aided drug design procedures can be resourcefully applied for the rapid identification of promising drug candidates. In the present study, we applied the molecular docking method in conjunction with molecular dynamics (MD) simulations to find out potential agonists for FXR based on structural similarity with the drug that is currently used as FXR agonist, obeticholic acid. Our results showed that alvimopan and montelukast could be used as potent FXR activators and outperform the binding affinity of obeticholic acid by forming stable conformation with the protein in silico. However, further investigational studies and validations of the selected drugs are essential to figure out their suitability for preclinical and clinical trials.     

Homology modeling of a novel epoxide hydrolase (EH) from Aspergillus niger SQ-6: structure-activity relationship in expoxides inhibiting EH activity

The 3D structure of a novel epoxide hydrolase from Aspergillus niger SQ-6 (sqEH) was constructed by using homology modeling and molecular dynamics simulations. Based on the 3D model, Asp191, His369 and Glu343 were predicted as catalytic triad. The putative active pocket is a hydrophobic environment and is rich in some important non—polar residues (Pro318, Trp282, Pro319, Pro317 and Phe242). Using three sets of epoxide inhibitors for docking study, the interaction energies of sqEH with each inhibitor are consistent with their inhibitory effects in previous experiments. Moreover, a critical water molecule which closes to the His369 was identified to be an ideal position for the hydrolysis step of the reaction. Two tyrosine residues (Tyr249 and Tyr312) are able to form hydrogen bonds with the epoxide oxygen atom to maintain the initial binding and positioning of the substrate in the active pocket. These docked complex models can well interpret the substrate specificity of sqEH, which could be relevant for the structural—based design of specific epoxide inhibitors. Figure       

Role of E542 and E545 missense mutations of PIK3CA in breast cancer: a comparative computational approach

Recent statistics describe breast cancer as the leading cause of death among women across the world with varied causes and reasons. Lifestyle, diet, genetic and environmental factors introduce their generous contributions towards breast cancer, among which genetic factors have lately become one of the most important aspects in understanding the mechanism. Although various genes have already been reported in causing breast cancer, PIK3CA stands second on the list. Mutations observed in this gene have the ability to trigger the different activities of the cell, thereby bypassing the regular cellular cycle. Among the mutations in PIK3CA, three hotspot mutations were commonly reported, one in the catalytic domain (position HIS1047) and other two in the helical domain (position GLU542 and GLU545). In the helical domain of PIK3CA, the lysine substitution at 542–545 positions was significantly studied in causing breast cancer. To compare the deleterious effect of these mutations, in silico prediction tools along with molecular dynamics simulations and molecular docking approach was initiated to analyse the change in binding landscape upon mutation. In this comparative analysis, we report that the mere existence of mutant E545K can trigger the function of the protein but may not be as harmful as H1047R. Among the two mutations E542K and E545K, the latter shows the most deleterious effect that correlates with the previous reported experimental studies. We assume the results observed in this combinatorial computational study might further pave a better way for providing better treatment procedures.     

Understanding the effects of two bound glucose in Sudlow site I on structure and function of human serum albumin: theoretical studies

Human serum albumin (HSA) is the most abundant protein found in blood serum. It carries essential metabolites and many drugs. The glycation of HSA causes abnormal biological effects. Importantly, glycated HSA (GHSA) is of interest as a biomarker for diabetes. Recently, the first HSA structure with bound pyranose (GLC) and open-chain (GLO) glucose at Sudlow site I has been crystallised. We therefore employed molecular dynamics (MD) simulations and ONIOM calculations to study the dynamic nature of two bound glucose in a pre-glycated HSA (pGHSA) and observe how those sugars alter a protein structure comparing to wild type (Apo) and fatty acid-bound HSA (FA). Our analyses show that the overall structural stability of pGHSA is similar to Apo and FA, except Sudlow site II. Having glucose induces large protein flexibility at Sudlow site II. Besides, the presence of glucose causes W214 to reorient resulting in a change in W214 microenvironment. Considering sugars, both sugars are exposed to water, but GLO is more solvent-accessible. ONIOM results show that glucose binding is favoured for HSA (?115.04 kcal/mol) and GLO (?85.10 kcal/mol) is more preferable for Sudlow site I over GLC (?29.94 kcal/mol). GLO can strongly react with K195 and K199, whereas K195 and K199 provide slightly repulsive forces for GLC. This can confirm that an open-chain GLO is more favourable inside a pocket.     

Calmodulin structure and function: Implication of arginine in the compaction related to ligand binding

Calmodulin (CAM) is a modulatory protein that regulates cellular activity by binding to a large number of proteins. Key elements in the Ca²⁺-dependent mechanism of interaction between CAM and the proteins it activates are the selectivity for Ca²⁺ ions and the requirement for Ca²⁺-dependent conformational changes. We report on results from a series of molecular dynamics simulations that identified discrete steps in the mechanism of structural rearrangement of CAM. The findings implicate the side chains of arginine residues in the bending of the central alpha helix. Structural and energetic considerations point to a dynamic hydrogen bonding pattern around the arginine residues as a ratcheting-type mechanism, causing the kinking of the central helix in consecutive steps stabilized by each new pattern of hydrogen bonds. Initial model building studies to locate potential binding sites of ligands such as trifluoperazine (TFP) indicate that the compaction of CAM results in several structural changes, that explain the selective binding of molecules such as TFP in the N-terminal domain. The present studies identify specific residues involved in the process of compaction and point to specific CAM residues involved in the binding of the ligand. These insights lead directly to propositions for experimental engineering of the molecular structure of CAM in order to probe the hypotheses and their consequences for the function of this important protein.     

Metabolic pathway analysis and molecular docking analysis for identification of putative drug targets in Toxoplasma gondii: novel approach

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that can infect a wide range of warm-blooded animals including humans. In humans and other intermediate hosts, toxoplasma develops into chronic infection that cannot be eliminated by host's immune response or by currently used drugs. In most cases, chronic infections are largely asymptomatic unless the host becomes immune compromised. Thus, toxoplasma is a global health problem and the situation has become more precarious due to the advent of HIV infections and poor toleration of drugs used to treat toxoplasma infection, having severe side effects and also resistance have been developed to the current generation of drugs. The emergence of these drug resistant varieties of T. gondii has led to a search for novel drug targets. We have performed a comparative analysis of metabolic pathways of the host Homo sapiens and the pathogen T. gondii. The enzymes in the unique pathways of T. gondii, which do not show similarity to any protein from the host, represent attractive potential drug targets. We have listed out 11 such potential drug targets which are playing some important work in more than one pathway. Out of these, one important target is Glutamate dehydrogenase enzyme; it plays crucial part in oxidation reduction, metabolic process and amino acid metabolic process. As this is also present in the targets of tropical diseases of TDR (Tropical disease related Drug) target database and no PDB and MODBASE 3D structural model is available, homology models for Glutamate dehydrogenase enzyme were generated using MODELLER9v6. The model was further explored for the molecular dynamics simulation study with GROMACS, virtual screening and docking studies with suitable inhibitors against the NCI diversity subset molecules from ZINC database, by using AutoDock-Vina. The best ten docking solutions were selected (ZINC01690699, ZINC17465979, ZINC17465983, ZINC18141294_03, ZINC05462670, ZINC01572309, ZINC18055497_01, ZINC18141294, ZINC05462674 and ZINC13152284_01). Further the Complexes were analyzed through LIGPLOT. On the basis of Complex scoring and binding ability it is deciphered that these NCI diversity set II compounds, specifically ZINC01690699 (as it has minimum energy score and one of the highest number of interactions with the active site residue), could be promising inhibitors for T. gondii using Glutamate dehydrogenase as Drug target.     

CD4-gp120 interaction interface - a gateway for HIV-1 infection in human: molecular network,modeling and docking studies

The major causative agent for Acquired Immune Deficiency Syndrome (AIDS) is Human Immunodeficiency Virus-1 (HIV-1). HIV-1 is a predominant subtype of HIV which counts on human cellular mechanism virtually in every aspect of its life cycle. Binding of viral envelope glycoprotein-gp120 with human cell surface CD4 receptor triggers the early infection stage of HIV-1. This study focuses on the interaction interface between these two proteins that play a crucial role for viral infectivity. The CD4–gp120 interaction interface has been studied through a comprehensive protein–protein interaction network (PPIN) analysis and highlighted as a useful step towards identifying potential therapeutic drug targets against HIV-1 infection. We prioritized gp41, Nef and Tat proteins of HIV-1 as valuable drug targets at early stage of viral infection. Lack of crystal structure has made it difficult to understand the biological implication of these proteins during disease progression. Here, computational protein modeling techniques and molecular dynamics simulations were performed to generate three-dimensional models of these targets. Besides, molecular docking was initiated to determine the desirability of these target proteins for already available HIV-1 specific drugs which indicates the usefulness of these protein structures to identify an effective drug combination therapy against AIDS.     

Structure and activity of membrane receptors: Modeling and computational simulation of ligand recognition in a three-dimensional model of the 5-hydroxytryptamine1A receptor

A three-dimensional molecular model of the transmembrane domain of the 5-HT_1A receptor (5-HT_1AR) is presented in the context of a general strategy for modeling the macromolecular structure of a guanine nucleotide binding, regulatory protein coupled receptor (GPCR). The model of the 5-HT_1AR rests on the definition of the putative residues of the ligand-binding site guided by criteria based on specific models proposed from structure-activity studies and on published results of modifications of GPCRs using methods of molecular biology. The resulting requirements for matching recognition sites in the agonist-binding pocket define the molecular details of the interaction between the agonist 5-HT and the human 5-HT_1AR that includes: (1) the interaction between the protonated amine moiety and the conserved negative Asp-116, located in TMH 3; (2) the hydrogen bond between the hydroxyl group and Thr-199, located in TMH 5; and (3) the interaction complex between the aromatic ring portion of the ligand and the neutral form of His-192, located in TMH 5. Results from quantum mechanical calculations of the interaction between an agonist and the proposed recognition pocket of the 5-HT_1AR model suggest a trigger of the receptor activation mechanism resulting from ligand binding. The antagonist-binding pocket of the human 5-HT_1AR is inferred from the interaction sites of pindolol with the receptor model: (1) the ionic interaction between the protonated amine of the ligand and the side chain of the conserved Asp-116, located in TMH 3; and (2) the hydrogen bonds between the ether oxygen and the hydroxyl group of the ligand and Asn-385, located in TMH 7. Use of the model is proposed to facilitate the identification of the structural elements of agonists and antagonists that are key for their specific functions, in order to achieve the design of new compounds with predetermined pharmacological properties.     

Opening of the ADP-bound active site in the ABC transporter ATPase dimer: evidence for a constant contact, alternating sites model for the catalytic cycle

ABC transporters are ubiquitous, ATP-dependent transmembrane pumps. The mechanism by which ATP hydrolysis in the nucleotide-binding domain (NBD) effects conformational changes in the transmembrane domain that lead to allocrite translocation remains largely unknown. A possible aspect of this mechanism was suggested by previous molecular dynamics simulations of the MJ0796 NBD dimer, which revealed a novel, nucleotide-dependent intrasubunit conformational change involving the relative rotation of the helical and catalytic subdomains. Here, we find that in four of five simulations of the ADP/ATP-bound dimer, the relative rotation of the helical and catalytic subdomains in the ADP-bound monomer results in opening of the ADP-bound active site, probably sufficient or close to sufficient to allow nucleotide exchange. We also observe that in all five simulations of the ADP/ATP-bound dimer, the intimate contact of the LSGGQ signature sequence with the ATP gamma-phosphate is weakened by the intrasubunit conformational change within the ADP-bound monomer. We discuss how these results support a constant contact model for the function of the NBD dimer in contrast to switch models, in which the NBDs are proposed to fully disassociate during the catalytic cycle.     

Multiple mapping method: a novel approach to the sequence-to-structure alignment problem in comparative protein structure modeling

A major bottleneck in comparative protein structure modeling is the quality of input alignment between the target sequence and the template structure. A number of alignment methods are available, but none of these techniques produce consistently good solutions for all cases. Alignments produced by alternative methods may be superior in certain segments but inferior in others when compared to each other; therefore, an accurate solution often requires an optimal combination of them. To address this problem, we have developed a new approach, Multiple Mapping Method (MMM). The algorithm first identifies the alternatively aligned regions from a set of input alignments. These alternatively aligned segments are scored using a composite scoring function, which determines their fitness within the structural environment of the template. The best scoring regions from a set of alternative segments are combined with the core part of the alignments to produce the final MMM alignment. The algorithm was tested on a dataset of 1400 protein pairs using 11 combinations of two to four alignment methods. In all cases MMM showed statistically significant improvement by reducing alignment errors in the range of 3 to 17%. MMM also compared favorably over two alignment meta-servers. The algorithm is computationally efficient; therefore, it is a suitable tool for genome scale modeling studies.     

Insect distribution in response to climate change based on a model: Review of function and use of CLIMEX

The significant dependence of agricultural productivity on pest control requires pest distribution predictions at an early stage of pest invasion. Because pest cycles are critically affected by climate, climate is one of the most important factors for predicting an invasive pest. CLIMEX is a highly effective tool that can predict potential geographical species distributions, and test the regional suitability for a target species' habitat based on data including climate change scenarios. CLIMEX has been recently used in Europe, North America, China and Australia, among others. However, for modeling species distributions in Korea, the use of the model has been limited to date. This study aimed to first introduce the function and application of CLIMEX by reviewing important studies using this model. Second, we investigated previous studies using the model simulation to demonstrate the practical applicability of CLIMEX for the agricultural sector, and its use in forecasting.     

An indirect approach to imply trade-off shapes: population level patterns in resistance suggest a decreasingly costly resistance mechanism in a model insect system

Trade-offs between life history and other traits play a key role in shaping the evolution of individuals. It is well established theoretically that the shapes of trade-off curves are as crucial to the evolutionary outcome as their strengths. However, measuring the shape of these relationships directly is often impractical. Here we use an indirect approach that examines the patterns seen within a population and then use theory to infer the shape of the trade-off curve. Using a bioassay we found that most individuals had either high susceptibility or relatively high resistance to a microparasite in a lepidopteran host population. According to general theory, this type of pattern in resistance would be most likely with a deceleratingly costly impact on fitness of increasing resistance. The implications and generality of the approach are discussed, along with the implications of the results to our understanding of the nature of innate resistance to parasites.     

Dynamics of a benzo[a]pyrene-derived guanine DNA lesion in TGT and CGC sequence contexts: enhanced mobility in TGT explains conformational heterogeneity, flexible bending, and greater susceptibility to nucleotide excision repair

The nucleotide excision repair (NER) machinery excises a variety of bulky DNA lesions, but with varying efficiencies. The structural features of the DNA lesions that govern these differences are not well understood. An intriguing model system for studying structure-function relationships in NER is the major adduct derived from the reaction of the highly tumorigenic metabolite of benzo[a]pyrene, (+)-anti-benzo[a]pyrene diol epoxide, with the exocyclic amino group of guanine ((+)-trans-anti-[BP]-N²-dG, or G*). The rates of incision of the stereochemically identical lesions catalyzed by the prokaryotic UvrABC system was shown to be greater by a factor of 2.3 ± 0.3 in the TG*T than in the CG*C sequence context [Biochemistry 46 (2007) 7006-7015]. Here we employ molecular dynamics simulations to elucidate the origin of the greater excision efficiency in the TG*T case and, more broadly, to delineate structural parameters that enhance NER. Our results show that the BP aromatic ring system is 5′-directed along the modified strand in the B-DNA minor groove in both sequence contexts. However, the TG*T modified duplex is much more dynamically flexible, featuring more perturbed and mobile Watson-Crick hydrogen bonding adjacent to the lesion, a greater impairment in stacking interactions, more dynamic local roll/bending, and more minor groove flexibility. These characteristics explain a number of experimental observations concerning the (+)-trans-anti-[BP]-N²-dG adduct in double-stranded DNA with the TG*T sequence context: its conformational heterogeneity in NMR solution studies, its highly flexible bend, and its lower thermal stability. By contrast, the CG*C modified duplex is characterized by a single BP conformation and a rigid bend. While current recognition models of bulky lesions by NER factors have stressed the importance of impaired Watson-Crick pairing/stacking and bending, our results highlight the likelihood of an important role for the local dynamics in the vicinity of the lesion.