Homology modeling and molecular dynamics simulations of the glycine receptor ligand binding domain

We present a homology based model of the ligand binding domain (LBD) of the homopentameric alpha1 glycine receptor (GlyR). The model is based on multiple sequence alignment with other members of the nicotinicoid ligand gated ion channel superfamily and two homologous acetylcholine binding proteins (AChBP) from the freshwater (Lymnaea stagnalis) and saltwater (Aplysia californica) snails with known high resolution structure. Using two template proteins with known structure to model three dimensional structure of a target protein is especially advantageous for sequences with low homology as in the case presented in this paper. The final model was cross-validated by critical evaluation of experimental and published mutagenesis, functional and other biochemical studies. In addition, a complex structure with strychnine antagonist in the putative binding site is proposed based on docking simulation using Autodock program. Molecular dynamics (MD) simulations with simulated annealing protocol are reported on the proposed LBD of GlyR, which is stable in 5 ns simulation in water, as well as for a deformed LBD structure modeled on the corresponding domain determined in low-resolution cryomicroscopy structure of the alpha subunit of the full-length acetylcholine receptor (AChR). Our simulations demonstrate that the beta-sandwich central core of the protein monomer is fairly rigid in the simulations and resistant to deformations in water.     

Interdomain dynamics and ligand binding: molecular dynamics simulations of glutamine binding protein

Periplasmic binding proteins from Gram-negative bacteria possess a common architecture, comprised of two domains linked by a hinge region, a fold which they share with the neurotransmitter-binding domains of ionotropic glutamate receptors (GluRs). Glutamine-binding protein (GlnBP) is one such protein, whose crystal structure has been solved in both open and closed forms. Multi-nanosecond molecular dynamics simulations have been used to explore motions about the hinge region and how they are altered by ligand binding. Glutamine binding is seen to significantly reduce inter-domain motions about the hinge region. Essential dynamics analysis of inter-domain motion revealed the presence of both hinge-bending and twisting motions, as has been reported for a related sugar-binding protein. Significantly, the influence of the ligand on GlnBP dynamics is similar to that previously observed in simulations of rat glutamate receptor (GluR2) ligand-binding domain. The essential dynamics analysis of GlnBP also revealed a third class of motion which suggests a mechanism for signal transmission in GluRs.     

Computer-aided de novo ligand design and docking/molecular dynamics study of Vitamin D receptor agonists

1α,25(OH)₂D₃, which is directly mediated by the vitamin D receptor (VDR), exerts a wide variety of biological actions. However, the treatment with 1α,25(OH)₂D₃ is limited because of its side effects. Many analogs and several nonsteroidal mimics with potent biological activity have been reported so far, and our rationale for designing the VDR agonists was on the basis of computer-aided drug design method by de novo design of A-ring and C/D-ring position of 1α,25(OH)₂D₃. Pyrimidine-2,4-diamine was selected as A-ring, and naphthalene and benzene were chosen as C/D-ring. By linking different components, a virtue compound library was obtained. To evaluate the contribution to activity of each component, we performed a series of automated molecular docking operations. Results revealed that the 19-dimethyl derivatives (the C-19 position correspond to C-20 in 1α,25(OH)₂D₃) show the favorable docking affinity to VDR. Moreover, the docking results are quite robust when further validated by molecular dynamics simulations. In addition, by free energy analysis using molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method, the driving force of the binding between VDR and the ligands is proved to be hydrophobic interactions. Thus, a possible strategy to design new series of VDR agonists is proposed. The strategy can be successfully applied to explain the high potential activities of the 19-dimethyl derivatives. It is anticipated that the findings reported here may provide useful information for designing effective VDR agonists as well as the therapeutic treatment of VDR-related diseases.     

Characterization of ligand binding,DNA binding and phosphorylation of progesterone receptor by two novel progesterone receptor antagonist ligands

In order to gain a better understanding of the distinctive mechanisms of the various types of antiprogestins, we have characterized in vitro ligand binding, specific DNA binding and phosphorylation of progesterone receptor (PR) from T47D cells after treatment of cells with progestins (progesterone, R5020) and antiprogestins (RU486, ZK98299, Org 31806 and Org 31710). Treatment of the cells with R5020 or PR antagonists, with the exception of ZK98299, resulted in a quantitative upshift of PR-A and PR-B indicative of ligand/DNA-induced phosphorylation of PR. Treatment of cells with RU486, Org 31710 or Org 31806, but not R5020 or ZK98299 resulted in detectable PR-progesterone response element complexes (PR-PREc) as assessed by gel mobility shift assay. Although treatment of cells with ZK98299, a type I PR antagonist, did not induce phosphorylation, the antiprogestins, Org 31806 and Org 31710, in a manner identical to RU486, did. Our data suggest that Org 31806 and Org 31710 affect propertie s of PR from T47D cells that are similar to RU486. (Mol Cell Biochem 175: 205–212, 1997)     

Biological activity and ligand binding mode to the progesterone receptor of A-homo analogues of progesterone

The biological activity of two seven-membered A-ring (A-homo) analogues of progesterone was evaluated by transactivation assays in Cos-1 cells and by determination of Bcl-x(L) expression levels in T47D cells. The results show that both compounds act as selective progesterone receptor (PR) agonists but lack mineralocorticoid receptor (MR) activity. Molecular modelling using semiempirical AM1 and ab initio HF/6-31G** calculations, showed that the A-ring of the A-homo steroids may adopt five different conformations, although only three correspond to low energy conformers. The low energy conformers of each analogue were introduced into the ligand binding pocket of the PR ligand binding domain (LBD) obtained from the PR LBD-progesterone crystal structure. The steroid binding mode was then analyzed using 10 ns of molecular dynamics (MD) simulation. The PR LBD-progesterone complex was also simulated as a control system. The MD results showed that both A-homo steroids have one conformer that may be properly recognized by the PR, in agreement with the observed progestagen activity. Moreover, the simulation revealed the importance of a water molecule in the formation of a hydrogen bonding network among specific receptor residues and the steroid A-ring carbonyl.     

Conformational dynamics of RNA-peptide binding: a molecular dynamics simulation study

Molecular dynamics simulations of the binding of the heterochiral tripeptide KkN to the transactivation responsive (TAR) RNA of HIV-1 is presented, using an all-atom force field with explicit water. To obtain starting structures for the TAR-KkN complex, semirigid docking calculations were performed that employ an NMR structure of free TAR RNA. The molecular dynamics simulations show that the starting structures in which KkN binds to the major groove of TAR (as it is the case for the Tat-TAR complex of HIV-1) are unstable. On the other hand, the minor-groove starting structures are found to lead to several binding modes, which are stabilized by a complex interplay of stacking, hydrogen bonding, and electrostatic interactions. Although the ligand does not occupy the binding position of Tat protein, it is shown to hinder the interhelical motion of free TAR RNA. The latter is presumably necessary to achieve the conformational change of TAR RNA to bind Tat protein. Considering the time evolution of the trajectories, the binding process is found to be ligand-induced and cooperative. That is, the conformational rearrangement only occurs in the presence of the ligand and the concerted motion of the ligand and a large part of the RNA binding site is necessary to achieve the final low-energy binding state.     

Exploring the role of the phospholipid ligand in endothelial protein C receptor: A molecular dynamics study

Endothelial protein C receptor (EPCR) is a CD1‐like transmembrane glycoprotein with important regulatory roles in protein C (PC) pathway, enhancing PC's anticoagulant, anti‐inflammatory, and antiapoptotic activities. Similarly to homologous CD1d, EPCR binds a phospholipid [phosphatidylethanolamine (PTY)] in a groove corresponding to the antigen‐presenting site, although it is not clear if lipid exchange can occur in EPCR as in CD1d. The presence of PTY seems essential for PC γ‐carboxyglutamic acid (Gla) domain binding. However, the lipid‐free form of the EPCR has not been characterized. We have investigated the structural role of PTY on EPCR, by multiple molecular dynamics (MD) simulations of ligand bound and unbound forms of the protein. Structural changes, subsequent to ligand removal, led to identification of two stable and folded ligand‐free conformations. Compared with the bound form, unbound structures showed a narrowing of the A′ pocket and a high flexibility of the helices around it, in agreement with CD1d simulation. Thus, a lipid exchange with a mechanism similar to CD1d is proposed. In addition, unbound conformations presented a reduced interaction surface for Gla domain, confirming the role of PTY in establishing the proper EPCR conformation for the interaction with its partner protein. Single MD simulations were also obtained for 29 mutant models with predicted structural stability and impaired binding ability. Ligand affinity calculations, based on linear interaction energy method, showed that substitution‐induced conformational changes affecting helices around the A′ pocket were associated to a reduced binding affinity. Mutants responsible for this effect may represent useful reagents for experimental tests. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Juxtaposition of the steroid binding domain-like I and II regions constitutes a ligand binding site in the sigma-1 receptor

sigma-1 receptors represent unique binding sites that are capable of interacting with a wide range of compounds to mediate different cellular events. The composition of the ligand binding site of this receptor is unclear, since no NMR or crystal structures are available. Recent studies in our laboratory using radiolabeled photoreactive ligands suggested that the steroid binding domain-like I (SBDLI) (amino acids 91-109) and the steroid binding domain-like II (SBDLII) (amino acids 176-194) regions are involved in forming the ligand binding site(s) ( Chen, Y., Hajipour, A. R., Sievert, M. K., Arbabian, M., and Ruoho, A. E. (2007) Biochemistry 46, 3532-3542 ; Pal, A., Hajipour, A. R., Fontanilla, D., Ramachandran, S., Chu, U. B., Mavlyutov, T., and Ruoho, A. E. (2007) Mol. Pharmacol. 72, 921-933 ). In this report, we have further addressed this issue by utilizing our previously developed sulfhydryl-reactive, cleavable, radioiodinated photocross-linking reagent: methanesulfonothioic acid, S-((4-(4-amino-3-[125I]iodobenzoyl) phenyl)methyl) ester (Guo, L. W., Hajipour, A. R., Gavala, M. L., Arbabian, M., Martemyanov, K. A., Arshavsky, V. Y., and Ruoho, A. E. (2005) Bioconjugate Chem. 16, 685-693). This photoprobe was shown to derivatize the single cysteine residues as mixed disulfides at position 94 in the SBDLI region of the wild type guinea pig sigma-1 receptor (Cys94) and at position 190 in the SBDLII region of a mutant guinea pig sigma-1 receptor (C94A,V190C), both in a sigma-ligand (haloperidol or (+)-pentazocine)-sensitive manner. Significantly, photocross-linking followed by Endo Lys-C cleavage under reducing conditions and intramolecular radiolabel transfer from the SBDLI to the SBDLII region in the wild type receptor and, conversely, from the SBDLII to the SBDLI region in the mutant receptor were observed. These data support a model in which the SBDLI and SBDLII regions are juxtaposed to form, at least in part, a ligand binding site of the sigma-1 receptor.     

The conformation of the extracellular binding domain of Death Receptor 5 in the presence and absence of the activating ligand TRAIL: a molecular dynamics study

The Death Receptor 5 (DR5), a member of tumor necrosis factor receptor (TNFR) superfamily of receptors, triggers apoptosis (programmed cell death) when stimulated by its tridentate ligand TRAIL. Until recently it was generally assumed that the activation of DR5 resulted from the recruitment of three independent receptor units, leading to the trimerization of intracellular domains. However, there is mounting evidence to suggest that, in the absence of ligand, such cytokine receptors primarily reside as preformed complexes. In this work, molecular dynamics simulations of the TRAIL-DR5 complex, the unbound receptor trimer and individual receptor monomers are compared to gain insight in the mechanism of activation. The results suggest that, in the absence of TRAIL, DR5 has a strong propensity to self-associate and that this is primarily mediated through interactions of the membrane proximal domains. The association of the free receptors leads to a loss of the threefold symmetry found within the receptor-ligand complex. The simulations suggest that the primary role of TRAIL is to induce threefold-symmetry within the DR5 complex and to constrain the receptor to a specific conformation. The implications of this in terms of the mechanism by which the receptor switches from an inactive to an active state are discussed.     

The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens

The human prostate tumor cell line LNCaP containd an abnormal androgen receptor system with broad steroid binding specificity. Progestagens, estradiol and several antiandrogens compete with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen sensitive systems. Optimal growth of LNCaP cells is observed after addition of the synthetic androgen R1881 (0.1 nM). In addition, estrogens, progestagens and several antiandrogens do not inhibit androgen responsive growth, but have striking growth stimulatory effects and increase EGF receptor level and acid phosphatase secretion. We have found that the androgen receptor in the LNCaP cells contains a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or HeLa cells. Androgens, progestagens, estrogens and several antiandrogens bind the mutated androgen receptor proteon and activate the expression of an androgen-regulated reporter gene (GRE-tk-CAT), indicating that the mutation directly affects both binding specificity and the induction of gene expression. Interestingly, the antiandrogen casodex showed antiandrogenic properties in growth studies of LNCaP cells and did not induced reporter gene activity in Hela cells transfected with the mutant receptor. The mutated androgen receptor of LNCaP cells is therefore a useful tool in the elucidation of different levels of action of steroids and antisteroids.     

Accurate detection of protein:ligand binding sites using molecular dynamics simulations

Accurate prediction of location of cavities and surface grooves in proteins is important, as these are potential sites for ligand binding. Several currently available programs for cavity detection are unable to detect cavities near the surface or surface grooves. In the present study, an optimized molecular dynamics based procedure is described for detection and quantification of interior cavities as well as surface pockets. This is based on the observation that the mobility of water in such pockets is significantly lower than that of bulk water. The algorithm efficiently detects surface grooves that are sites of protein-ligand and protein-protein interaction. The algorithm was also used to substantially improve the performance of an automated docking procedure for docking monomers of nonobligate protein-protein complexes. In addition, it was applied to predict key residues involved in the binding of the E. coli toxin CcdB with its inhibitor. Predictions were subsequently validated by mutagenesis experiments.     

Role of ligand binding in structural organization of add A-riboswitch aptamer: a molecular dynamics simulation

The specific binding of ligands is the first step of gene expression or translation regulation by riboswitches. However, understanding the mechanism of the specific binding is still difficult because the tertiary structures of the riboswitch aptamers are available almost only for ligand-bound state at present. In this paper we hope to give some insights into this problem through the studies of the role of ligand-aptamer interaction in the structural organization of add A-riboswitch aptamer, based on the crystal structure of the ligand-bound aptamer. We use all-atom molecular dynamics to simulate the behaviors of the aptamer in ligand-bound, free and mutated states by Amber force field. The results show that the correct paring of the ligand adenine with the nucleotide U74 in the binding pocket is crucial to stabilizing the conformations of the ligand-bound aptamer, especially the helix P1 connecting the expression platform. Our results also suggest that both the nucleotide U74 and U51 may be the key sites of the ligand recognition but the former has much higher probability as the initial docking site. This is in agreement with previous experimental results.     

The platelet fibrinogen receptor: an immunogold-surface replica study of agonist-induced ligand binding and receptor clustering

Platelet aggregation requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma-membrane glycoproteins (GP) IIb and IIIa. Although the GPIIb-IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold-surface replica technique to study the distribution of GPIIb-IIIa and bound fibrinogen over broad areas of surface membranes in unstimulated, as well as thrombin-activated and ADP-activated human platelets. We found that the immunogold-labeled GPIIb-IIIa was monodispersed over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. On thrombin-stimulated platelets, approximately 65% of the GPIIb-IIIa molecules were in clusters within the plane of the membrane. Fibrinogen, which had been released from the alpha-granules of these cells, bound to GPIIb-IIIa on the cell surface and was similarly clustered. To determine whether the receptors clustered before ligand binding, or as a consequence thereof, we studied the surface distribution of GPIIb-IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb-IIIa without inducing the release of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding-competent receptors on ADP-stimulated platelets were monodispersed. The addition of fibrinogen caused the GPIIb-IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb-IIIa-binding domains of fibrinogen, namely the tetrapeptide Arg-Gly-Asp-Ser on the alpha-chain or the gamma-chain decapeptide gamma 402-411. These results show that receptor occupancy causes clustering of GPIIb-IIIa in activated platelets.     

The design of potent HIV-1 integrase inhibitors by a combined approach of structure-based virtual screening and molecular dynamics simulation

Communicated by Ramaswamy H. Sarma     

Asymmetric structural motions of the homomeric alpha7 nicotinic receptor ligand binding domain revealed by molecular dynamics simulation

A homology model of the ligand binding domain of the alpha7 nicotinic receptor is constructed based on the acetylcholine-binding protein crystal structure. This structure is refined in a 10 ns molecular dynamics simulation. The modeled structure proves fairly resilient, with no significant changes at the secondary or tertiary structural levels. The hypothesis that the acetylcholine-binding protein template is in the activated or desensitized state, and the absence of a bound agonist in the simulation suggests that the structure may also be relaxing from this state to the activatable state. Candidate motions that take place involve not only the side chains of residues lining the binding sites, but also the subunit positions that determine the overall shape of the receptor. In particular, two nonadjacent subunits move outward, whereas their partners counterclockwise to them move inward, leading to a marginally wider interface between themselves and an overall asymmetric structure. This in turn affects the binding sites, producing two that are more open and characterized by distinct side-chain conformations of W54 and L118, although motions of the side chains of all residues in every binding site still contribute to a reduction in binding site size, especially the outward motion of W148, which hinders acetylcholine binding. The Cys loop at the membrane interface also displays some flexibility. Although the short simulation timescale is unlikely to sample adequately all the conformational states, the pattern of observed motions suggests how ligand binding may correlate with larger-scale subunit motions that would connect with the transmembrane region that controls the passage of ions. Furthermore, the shape of the asymmetry with binding sites of differing affinity for acetylcholine, characteristic of other nicotinic receptors, may be a natural property of the relaxed, activatable state of alpha7.     

Modeling of the binding mode of a non-covalent inhibitor of the 20S proteasome. Application to structure-based analogue design

The 2-aminobenzvlstatine derivative I is a 20S proteasome inhibitor of a novel chemical type identified by high throughput screening. The compound specifically inhibits the chymotrypsin-like catalytic activity of the human proteasome with an IC50 value in the micromolar range. Using the crystal structure of the yeast proteasome, we modeled the structure of the human proteasome in complex with 1. As one of the first applications of the model in our oncology programme targeting the proteasome, we designed an analogue of the inhibitor having enhanced stacking/hydrophobic interactions with the enzyme. One order of magnitude in inhibitory potency was gained.     

Trifluoromethyl group as a pharmacophore: effect of replacing a CF3 group on binding and agonist activity of a glucocorticoid receptor ligand

Compound 1, a potent glucocorticoid receptor ligand, contains a quaternary carbon bearing trifluoromethyl and hydroxyl groups. This paper describes the effect of replacing the trifluoromethyl group on binding and agonist activity of the GR ligand 1. The results illustrate that replacing the CF3 group with a cyclohexylmethyl or benzyl group maintains the GR binding potency. These substitutions alter the functional behavior of the GR ligands from agonists to antagonists. Docking studies suggest that the benzyl analog 19 binds in a similar fashion as the GR antagonist, RU486. The central benzyl group of 19 and the C-11 dimethylaniline moiety of RU486 overlay. Binding of compound 19 is believed to force helix 12 to adopt an open conformation and this leads to the antagonist properties of the non-CF3 ligands carrying a large group at the center of the molecule.     

The role of tyrosine in the binding of steroid by progesterone binding globulin from the guinea pig

Treatment of progesterone binding globulin (PBG) with tetranitromethane (TNM) resulted in a loss of steroid binding activity (inactivation) which was dependent on both time and concentration of reagent. Scatchard analysis of binding revealed that inactivation was due to a decrease in binding site number with no effect upon the affinity of PBG for steroid. Incorporation studies demonstrated that the loss of binding activity correlated with the incorporation of 1.3 nitro groups per molecule of PBG. The involvement of the steroid binding site in the reaction was shown by the ability of progesterone, but not cortisol, to protect against inactivation. Treatment with N-acetylimidazole did not inactivate PBG nor did the conversion of nitrotyrosyl residues to amino-tyrosines regenerate binding activity, suggesting that the pheolic hydroxyl is not involved in steroid binding. These studies suggest that inactivation was due to the incorporation of a bulky group into the aromatic ring of a tyrosine present at the steroid binding site thus blocking its ability to participate in hydrophobic interactions with the ligand.