A functional selectivity mechanism at the serotonin-2A GPCR involves ligand-dependent conformations of intracellular loop 2.

With recent progress in determination of G protein-coupled receptor (GPCR) structure with crystallography, a variety of other experimental approaches (e.g., NMR spectroscopy, fluorescent-based assays, mass spectrometry techniques) are also being used to characterize state-specific and ligand-specific conformational states. MD simulations offer a powerful complementary approach to elucidate the dynamic features associated with ligand-specific GPCR conformations. To shed light on the conformational elements and dynamics of the important aspect of GPCR functional selectivity, we carried out unbiased microsecond-length MD simulations of the human serotonin 2A receptor (5-HT(2A)R) in the absence of ligand and bound to four distinct serotonergic agonists. The 5-HT(2A)R is a suitable system to study the structural features involved in the ligand-dependent conformational heterogeneity of GPCRs because it is well-characterized experimentally and exhibits a strong agonist-specific phenotype in that some 5-HT(2A)R agonists induce LSD-like hallucinations, while others lack this psychoactive property entirely. Here we report evidence for structural and dynamic differences in 5-HT(2A)R interacting with such pharmacologically distinct ligands, hallucinogens, and nonhallucinogens obtained from all-atom MD simulations. Differential ligand binding contacts were identified for structurally similar hallucinogens and nonhallucinogens and found to correspond to different conformations in the intracellular loop 2 (ICL2). From the different ICL2 conformations, functional selective phenotypes are suggested through effects on dimerization and/or distinct direct interaction with effector proteins. The findings are presented in the context of currently proposed hallucinogenesis mechanisms, and ICL2 is proposed as a fine-tuning selective switch that can differentiates modes of 5-HT(2A)R activation.

Beta cyclodextrins bind, stabilize, and remove lipofuscin bisretinoids from retinal pigment epithelium.

Accumulation of lipofuscin bisretinoids (LBs) in the retinal pigment epithelium (RPE) is the alleged cause of retinal degeneration in genetic blinding diseases (e.g., Stargardt) and a possible etiological agent for age-related macular degeneration. Currently, there are no approved treatments for these diseases; hence, agents that efficiently remove LBs from RPE would be valuable therapeutic candidates. Here, we show that beta cyclodextrins (ß-CDs) bind LBs and protect them against oxidation. Computer modeling and biochemical data are consistent with the encapsulation of the retinoid arms of LBs within the hydrophobic cavity of ß-CD. Importantly, ß-CD treatment reduced by 73% and 48% the LB content of RPE cell cultures and of eyecups obtained from Abca4-Rdh8 double knock-out (DKO) mice, respectively. Furthermore, intravitreal administration of ß-CDs reduced significantly the content of bisretinoids in the RPE of DKO animals. Thus, our results demonstrate the effectiveness of ß-CDs to complex and remove LB deposits from RPE cells and provide crucial data to develop novel prophylactic approaches for retinal disorders elicited by LBs.

Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation.

The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward- and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward- and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT undergoes significant conformational changes in transitions necessary for both cocaine binding and substrate transport.

Virtual ligand screening combined with NMR to identify Dvl PDZ domain inhibitors targeting the Wnt signaling.

Virtual ligand screening is a powerful technique to identify potential hits of targets and to increase hit rates. Here, we describe how we used this technique combined with NMR (15)N HSQC experiments to obtain small molecules that bind to the PDZ domain of Dvl targeting the Wnt signaling pathway.

Chemical and genetic evidence for the involvement of Wnt antagonist Dickkopf2 in regulation of glucose metabolism.

Mutations in Wnt receptor LRP5/6 and polymorphism in Wnt-regulated transcription factor TCF7L2 are associated with dysregulation of glucose metabolism. However, it is not clear whether Wnt antagonist Dickkopf (Dkk) has a significant role in the regulation of glucose metabolism. Here, we identified small-molecule inhibitors of Wnt antagonist Dkk through molecular modeling, computation-based virtual screens, and biological assays. One of the Dkk inhibitors reduced basal blood-glucose concentrations and improved glucose tolerance in mice. This Dkk inhibitor appeared to act through DKK2 because the inhibitor exerted no additional effects on glucose metabolism in the Dkk2(-/-) mice. Our study of Dkk2(-/-) mice showed that DKK2 deficiency was associated with increased hepatic glycogen accumulation and decreased hepatic glucose output. DKK2 deficiency did not cause in increase in insulin production but resulted in increased Wnt activity and GLP1 production in the intestines. Given that the Dkk inhibitor improved glucose tolerance in a murine model of type 2 diabetes (db/db), we suggest that DKK2 may be a potential therapeutic target for treating type 2 diabetes.

Ligand-dependent conformations and dynamics of the serotonin 5-HT(2A) receptor determine its activation and membrane-driven oligomerization properties.

From computational simulations of a serotonin 2A receptor (5-HT(2A)R) model complexed with pharmacologically and structurally diverse ligands we identify different conformational states and dynamics adopted by the receptor bound to the full agonist 5-HT, the partial agonist LSD, and the inverse agonist Ketanserin. The results from the unbiased all-atom molecular dynamics (MD) simulations show that the three ligands affect differently the known GPCR activation elements including the toggle switch at W6.48, the changes in the ionic lock between E6.30 and R3.50 of the DRY motif in TM3, and the dynamics of the NPxxY motif in TM7. The computational results uncover a sequence of steps connecting these experimentally-identified elements of GPCR activation. The differences among the properties of the receptor molecule interacting with the ligands correlate with their distinct pharmacological properties. Combining these results with quantitative analysis of membrane deformation obtained with our new method (Mondal et al, Biophysical Journal 2011), we show that distinct conformational rearrangements produced by the three ligands also elicit different responses in the surrounding membrane. The differential reorganization of the receptor environment is reflected in (i)-the involvement of cholesterol in the activation of the 5-HT(2A)R, and (ii)-different extents and patterns of membrane deformations. These findings are discussed in the context of their likely functional consequences and a predicted mechanism of ligand-specific GPCR oligomerization.

Synthesis of potent dishevelled PDZ domain inhibitors guided by virtual screening and NMR studies.

Dishevelled (Dvl) PDZ domains transduce Wnt signals from the membrane-bound receptor Frizzled to the downstream. As abnormal Wnt signaling has been implicated in tumorigenesis, the Dvl PDZ domain is a potential target for small-molecule inhibitors that block Wnt signaling at the Dvl level. We expanded our in silico search to examine the chemical space near previously developed PDZ binders and identified nine additional compounds bind to the Dvl PDZ. We then performed a quantitative structure-activity relationship (QSAR) analysis of these compounds and combined these results with structural studies of the PDZ domain in complex with the compounds to design and synthesize a group of new, further optimized compounds. Two rounds of synthesis and testing yielded a total of six compounds that have greatly improved binding affinity to the Dvl PDZ domain and most potent ones competitively displace Dapper peptide from the PDZ domain. In addition to providing more potent Dvl PDZ domain inhibitors, this study demonstrates that virtual screening and structural studies can be powerful tools in guiding the chemical synthesis hit-to-lead optimization stage during the drug discovery process.

Quantitative modeling of membrane deformations by multihelical membrane proteins: application to G-protein coupled receptors.

The interpretation of experimental observations of the dependence of membrane protein function on the properties of the lipid membrane environment calls for a consideration of the energy cost of protein-bilayer interactions, including the protein-bilayer hydrophobic mismatch. We present a novel (to our knowledge) multiscale computational approach for quantifying the hydrophobic mismatch-driven remodeling of membrane bilayers by multihelical membrane proteins. The method accounts for both the membrane remodeling energy and the energy contribution from any partial (incomplete) alleviation of the hydrophobic mismatch by membrane remodeling. Overcoming previous limitations, it allows for radially asymmetric bilayer deformations produced by multihelical proteins, and takes into account the irregular membrane-protein boundaries. The approach is illustrated by application to two G-protein coupled receptors: rhodopsin in bilayers of different thickness, and the serotonin 5-HT(2A) receptor bound to pharmacologically different ligands. Analysis of the results identifies the residual exposure that is not alleviated by bilayer adaptation, and its quantification at specific transmembrane segments is shown to predict favorable contact interfaces in oligomeric arrays. In addition, our results suggest how distinct ligand-induced conformations of G-protein coupled receptors may elicit different functional responses through differential effects on the membrane environment.

Calculation of pK(a) in proteins with the microenvironment modulated-screened coulomb potential.

The MM-SCP has been applied to predict pK(a) values of titratable residues in wild type and mutants of staphylococcal nuclease (SNase). The calculations were based on crystal structures made available by the Garcia-Moreno Laboratory. In the mutants, mostly deeply buried hydrophobic residues were replaced with ionizable residues, and thus their pK(a) values could be measured and calculated using various methods. The data set used here consisted of a set of WT SNase for which His pK(a) at several ionic strengths had been measured, a set of mutants for which measured pK(a) were available and a set of 11 mutants for which the measured pK(a) were not known at the time of calculation. For this latter set, blind predictions were submitted to the protein pK(a) cooperative, 2009 workshop at Telluride, where the results of the blind predictions were discussed (the RMSD of the submitted set was 1.10 pH units). The calculations on the structures with known pK(a) indicated that in addition to weaknesses of the method, structural issues were observed that led to larger errors (>1) in pK(a) predictions. For example, different crystallographic conditions or steric clashes can lead to differences in the local environment around the titratable residue, which can produce large differences in the calculated pK(a) . To gain further insight into the reliability of the MM-SCP, pK(a) of an extended set of 54 proteins belonging to several structural classes were carried out. Here some initial results from this study are reported to help place the SNase results in the appropriate context.

The substrate-driven transition to an inward-facing conformation in the functional mechanism of the dopamine transporter.

BACKGROUND: The dopamine transporter (DAT), a member of the neurotransmitter:Na(+) symporter (NSS) family, terminates dopaminergic neurotransmission and is a major molecular target for psychostimulants such as cocaine and amphetamine, and for the treatment of attention deficit disorder and depression. The crystal structures of the prokaryotic NSS homolog of DAT, the leucine transporter LeuT, have provided critical structural insights about the occluded and outward-facing conformations visited during the substrate transport, but only limited clues regarding mechanism. To understand the transport mechanism in DAT we have used a homology model based on the LeuT structure in a computational protocol validated previously for LeuT, in which steered molecular dynamics (SMD) simulations guide the substrate along a pathway leading from the extracellular end to the intracellular (cytoplasmic) end. METHODOLOGY/PRINCIPAL FINDINGS: Key findings are (1) a second substrate binding site in the extracellular vestibule, and (2) models of the conformational states identified as occluded, doubly occupied, and inward-facing. The transition between these states involve a spatially ordered sequence of interactions between the two substrate-binding sites, followed by rearrangements in structural elements located between the primary binding site and the cytoplasmic end. These rearrangements are facilitated by identified conserved hinge regions and a reorganization of interaction networks that had been identified as gates. CONCLUSIONS/SIGNIFICANCE: Computational simulations supported by information available from experiments in DAT and other NSS transporters have produced a detailed mechanistic proposal for the dynamic changes associated with substrate transport in DAT. This allosteric mechanism is triggered by the binding of substrate in the S2 site in the presence of the substrate in the S1 site. Specific structural elements involved in this mechanism, and their roles in the conformational transitions illuminated here describe, a specific substrate-driven allosteric mechanism that is directly amenable to experiment as shown previously for LeuT.

Probing the structural determinants for the function of intracellular loop 2 in structurally cognate G-protein-coupled receptors.

Intracellular loop 2 (IL2) in G-protein-coupled receptors (GPCRs) is functionally important, e.g., in binding to G-protein and ß-arrestin. Differences in secondary structure of IL2 in the crystal structures of the very similar ß(1)- and ß(2)-adrenergic receptors (ß(1)AR and ß(2)AR, respectively), i.e., an α-helix and an L-shaped strand, respectively, emphasize the need to understand the structural basis for IL2 functionality. We studied the properties of IL2 in the context of experimental data using a Monte Carlo-based ab initio method. The procedure was validated first by verifying that the IL2 structures in ß(1)AR and ß(2)AR crystals were correctly reproduced, even after conformational ensemble searches at >1200 K where most secondary structure had been lost. We found that IL2 in ß(1)AR and ß(2)AR sampled each other's conformation but adopted different energetically preferred conformations, consistent with the crystal structures. The results indicate a persistent contextual preference for the structure of IL2, which was conserved when the IL2 sequences were interchanged between the receptors. We conclude that the protein environment, more than the IL2 sequence, regulates the IL2 structures. We extended the approach to the molecular model of 5-HT(2A)R for which no crystal structure is available and found that IL2 is predominantly helical, similar to IL2 in ß(1)AR. Because the P3.57A mutation in IL2 had been shown to decrease ß-arrestin binding and internalization, we predicted the effects of the mutation and found that it decreased the propensity of IL2 to form helix, identifying the helical IL2 as a component of the GPCR active form.

GPCR-OKB: the G Protein Coupled Receptor Oligomer Knowledge Base.

SUMMARY: Rapid expansion of available data about G Protein Coupled Receptor (GPCR) dimers/oligomers over the past few years requires an effective system to organize this information electronically. Based on an ontology derived from a community dialog involving colleagues using experimental and computational methodologies, we developed the GPCR-Oligomerization Knowledge Base (GPCR-OKB). GPCR-OKB is a system that supports browsing and searching for GPCR oligomer data. Such data were manually derived from the literature. While focused on GPCR oligomers, GPCR-OKB is seamlessly connected to GPCRDB, facilitating the correlation of information about GPCR protomers and oligomers. AVAILABILITY AND IMPLEMENTATION: The GPCR-OKB web application is freely available at http://www.gpcr-okb.org

Discovery and characterization of a small molecule inhibitor of the PDZ domain of dishevelled.

Dishevelled (Dvl) is an essential protein in the Wnt signaling pathways; it uses its PDZ domain to transduce the Wnt signals from the membrane receptor Frizzled to downstream components. Here, we report identifying a drug-like small molecule compound through structure-based ligand screening and NMR spectroscopy and show the compound to interact at low micromolar affinity with the PDZ domain of Dvl. In a Xenopus testing system, the compound could permeate the cell membrane and block the Wnt signaling pathways. In addition, the compound inhibited Wnt signaling and reduced the levels of apoptosis in the hyaloid vessels of eye. Moreover, this compound also suppressed the growth of prostate cancer PC-3 cells. These biological effects suggest that by blocking the PDZ domain of Dvl, the compound identified in our studies effectively inhibits the Wnt signaling and thus provides a useful tool for studies dissecting the Wnt signaling pathways.

Optimizing Dvl PDZ domain inhibitor by exploring chemical space.

Because of advances in the high-throughput screening technology, identification of a hit that can bind to a target protein has become a relatively easy task; however, in the process of drug discovery, the following hit-to-lead and lead optimization still remain challenging. In a typical hit-to-lead and lead optimization process, the analogues of the most promising hits are synthesized for the development of structure-activity relationship (SAR) analysis, and in turn, in the effort of optimization of lead compounds, such analysis provides guidance for the further synthesis. The synthesis processes are usually long and labor-intensive. In silico searching has becoming an alternative approach to explore SAR especially with millions of compounds ready to be screened and most of them can be easily obtained. Here, we report our discovery of 15 new Dishevelled PDZ domain inhibitors by using such an approach. In our studies, we first developed a pharmacophore model based on NSC668036, an inhibitor previously identified in our laboratory; based on the model, we then screened the ChemDiv database by using an algorithm that combines similarity search and docking procedures; finally, we selected potent inhibitors based on docking analysis and examined them by using NMR spectroscopy. NMR experiments showed that all the 15 compounds we chose bound to the PDZ domain tighter than NSC668036.

Functional insight into Maelstrom in the germline piRNA pathway: a unique domain homologous to the DnaQ-H 3'-5' exonuclease, its lineage-specific expansion/loss and evolutionarily active site switch.

Maelstrom (MAEL) plays a crucial role in a recently-discovered piRNA pathway; however its specific function remains unknown. Here a novel MAEL-specific domain characterized by a set of conserved residues (Glu-His-His-Cys-His-Cys, EHHCHC) was identified in a broad range of species including vertebrates, sea squirts, insects, nematodes, and protists. It exhibits ancient lineage-specific expansions in several species, however, appears to be lost in all examined teleost fish species. Functional involvement of MAEL domains in DNA- and RNA-related processes was further revealed by its association with HMG, SR-25-like and HDAC_interact domains. A distant similarity to the DnaQ-H 3'-5' exonuclease family with the RNase H fold was discovered based on the evidence that all MAEL domains adopt the canonical RNase H fold; and several protist MAEL domains contain the conserved 3'-5' exonuclease active site residues (Asp-Glu-Asp-His-Asp, DEDHD). This evolutionary link together with structural examinations leads to a hypothesis that MAEL domains may have a potential nuclease activity or RNA-binding ability that may be implicated in piRNA biogenesis. The observed transition of two sets of characteristic residues between the ancestral DnaQ-H and the descendent MAEL domains may suggest a new mode for protein function evolution called "active site switch", in which the protist MAEL homologues are the likely evolutionary intermediates due to harboring the specific characteristics of both 3'-5' exonuclease and MAEL domains.

Structural insight into the mechanisms of Wnt signaling antagonism by Dkk.

Dickkopf (Dkk) proteins are antagonists of the canonical Wnt signaling pathway and are crucial for embryonic cell fate and bone formation. Wnt antagonism of Dkk requires the binding of the C-terminal cysteine-rich domain of Dkk to the Wnt coreceptor, LRP5/6. However, the structural basis of the interaction between Dkk and low density lipoprotein receptor-related protein (LRP) 5/6 is unknown. In this study, we examined the structure of the Dkk functional domain and elucidated its interactions with LRP5/6. Using NMR spectroscopy, we determined the solution structure of the C-terminal cysteine-rich domain of mouse Dkk2 (Dkk2C). Then, guided by mutagenesis studies, we docked Dkk2C to the YWTD beta-propeller domains of LRP5/6 and showed that the ligand binding site of the third LRP5/6 beta-propeller domain matches Dkk2C best, suggesting that this domain binds to Dkk2C with higher affinity. Such differential binding affinity is likely to play an essential role in Dkk function in the canonical Wnt pathway.

An antagonist of dishevelled protein-protein interaction suppresses beta-catenin-dependent tumor cell growth.

Recent progress in the development of inhibitors of protein-protein interactions has opened the door for developing drugs that act by novel and selective mechanisms. Building on that work, we designed a small-molecule inhibitor of the Wnt signaling pathway, which is aberrantly activated across a wide range of human tumors. The compound, named FJ9, disrupts the interaction between the Frizzed-7 Wnt receptor and the PDZ domain of Dishevelled, down-regulating canonical Wnt signaling and suppressing tumor cell growth. The antitumorigenic effects of FJ9 were pronounced, including induction of apoptosis in human cancer cell lines and tumor growth inhibition in a mouse xenograft model. FJ9 is thus among the first non-peptide inhibitors to show therapeutic efficacy through disruption of PDZ protein-protein interactions.

Identification of a specific inhibitor of the dishevelled PDZ domain.

The Wnt signaling pathways are involved in embryo development as well as in tumorigenesis. Dishevelled (Dvl) transduces Wnt signals from the receptor Frizzled (Fz) to downstream components in canonical and noncanonical Wnt signaling pathways. The Dvl PDZ domain is thought to play an essential role in both pathways, and we recently demonstrated that the Dvl PDZ domain binds directly to Fz receptors. In this study, using structure-based virtual ligand screening, we identified an organic molecule (NSC668036) from the National Cancer Institute small-molecule library that can bind to the Dvl PDZ domain. We then used molecular dynamics simulation to analyze the binding between the PDZ domain and NSC668036 in detail. In addition, we showed that, in Xenopus, as expected, NSC668036 inhibited the signaling induced by Wnt3A. This compound provides a basis for rational design of high-affinity inhibitors of the PDZ domain, which can block Wnt signaling by interrupting the Fz-Dvl interaction.