Blockade of neuronal ?7-nAChR by ?-conotoxin ImI explained by computational scanning and energy calculations.
ABSTRACT: ?-Conotoxins potently inhibit isoforms of nicotinic acetylcholine receptors (nAChRs), which are essential for neuronal and neuromuscular transmission. They are also used as neurochemical tools to study nAChR physiology and are being evaluated as drug leads to treat various neuronal disorders. A number of experimental studies have been performed to investigate the structure-activity relationships of conotoxin/nAChR complexes. However, the structural determinants of their binding interactions are still ambiguous in the absence of experimental structures of conotoxin-receptor complexes. In this study, the binding modes of ?-conotoxin ImI to the ?7-nAChR, currently the best-studied system experimentally, were investigated using comparative modeling and molecular dynamics simulations. The structures of more than 30 single point mutants of either the conotoxin or the receptor were modeled and analyzed. The models were used to explain qualitatively the change of affinities measured experimentally, including some nAChR positions located outside the binding site. Mutational energies were calculated using different methods that combine a conformational refinement procedure (minimization with a distance dependent dielectric constant or explicit water, or molecular dynamics using five restraint strategies) and a binding energy function (MM-GB/SA or MM-PB/SA). The protocol using explicit water energy minimization and MM-GB/SA gave the best correlations with experimental binding affinities, with an R2 value of 0.74. The van der Waals and non-polar desolvation components were found to be the main driving force for binding of the conotoxin to the nAChR. The electrostatic component was responsible for the selectivity of the various ImI mutants. Overall, this study provides novel insights into the binding mechanism of ?-conotoxins to nAChRs and the methodological developments reported here open avenues for computational scanning studies of a rapidly expanding range of wild-type and chemically modified ?-conotoxins.
Project description:Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that belong to the superfamily of Cys loop receptors. Valuable insight into the orthosteric ligand binding to nAChRs in recent years has been obtained from the crystal structures of acetylcholine-binding proteins (AChBPs) that share significant sequence homology with the amino-terminal domains of the nAChRs. alpha-Conotoxins, which are isolated from the venom of carnivorous marine snails, selectively inhibit the signaling of neuronal nAChR subtypes. Co-crystal structures of alpha-conotoxins in complex with AChBP show that the side chain of a highly conserved proline residue in these toxins is oriented toward the hydrophobic binding pocket in the AChBP but does not have direct interactions with this pocket. In this study, we have designed and synthesized analogues of alpha-conotoxins ImI and PnIA[A10L], by introducing a range of substituents on the Pro(6) residue in these toxins to probe the importance of this residue for their binding to the nAChRs. Pharmacological characterization of the toxin analogues at the alpha(7) nAChR shows that although polar and charged groups on Pro(6) result in analogues with significantly reduced antagonistic activities, analogues with aromatic and hydrophobic substituents in the Pro(6) position exhibit moderate activity at the receptor. Interestingly, introduction of a 5-(R)-phenyl substituent at Pro(6) in alpha-conotoxin ImI gives rise to a conotoxin analogue with a significantly higher binding affinity and antagonistic activity at the alpha(7) nAChR than those exhibited by the native conotoxin.
Project description:Alpha-conotoxins are small disulfide-constrained peptides from cone snails that act as antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). The 13-residue peptide alpha-conotoxin RgIA (alpha-RgIA) is a member of the alpha-4,3 family of alpha-conotoxins and selectively blocks the alpha9alpha10 nAChR subtype, in contrast to another well-characterized member of this family, alpha-conotoxin ImI (alpha-ImI), which is a potent inhibitor of the alpha7 and alpha3beta2 nAChR subtypes. In this study, we have altered side chains in both the four-residue and the three-residue loops of alpha-RgIA, and have modified its C-terminus. The effects of these changes on activity against alpha9alpha10 and alpha7 nAChRs were measured; the solution structures of alpha-RgIA and its Y10W, D5E, and P6V analogues were determined from NMR data; and resonance assignments were made for alpha-RgIA [R9A]. The structures for alpha-RgIA and its three analogues were well defined, except at the chain termini. Comparison of these structures with reported structures of alpha-ImI reveals a common two-loop backbone architecture within the alpha-4,3 family, but with variations in side-chain solvent accessibility and orientation. Asp5, Pro6, and Arg7 in loop 1 are critical for blockade of both the alpha9alpha10 and the alpha7 subtypes. In loop 2, alpha-RgIA [Y10W] had activity near that of wild-type alpha-RgIA, with high potency for alpha9alpha10 and low potency for alpha7, and had a structure similar to that of wild type. By contrast, Arg9 in loop 2 is critical for specific binding to the alpha9alpha10 subtype, probably because it is larger and more solvent accessible than Ala9 in alpha-ImI. Our findings contribute to a better understanding of the molecular basis for antagonism of the alpha9alpha10 nAChR subtype, which is a target for the development of analgesics for the treatment of chronic neuropathic pain.
Project description:Nicotinic acetylcholine receptors (nAChRs) are targets for developing new drugs to treat severe pain, nicotine addiction, Alzheimer disease, epilepsy, etc. ?-Conotoxins are biologically and chemically diverse. With 12-19 residues and two disulfides, they can be specifically selected for different nAChRs. Acetylcholine-binding proteins from Aplysia californica (Ac-AChBP) are homologous to the ligand-binding domains of nAChRs and pharmacologically similar. X-ray structures of the ?-conotoxin in complex with Ac-AChBP in addition to computer modeling have helped to determine the binding site of the important residues of ?-conotoxin and its affinity for nAChR subtypes. Here, we present the various ?-conotoxin residues that are selective for Ac-AChBP or nAChRs by comparing the structures of ?-conotoxins in complex with Ac-AChBP and by modeling ?-conotoxins in complex with nAChRs. The knowledge of these binding sites will assist in the discovery and design of more potent and selective ?-conotoxins as drug leads.
Project description:Nicotinic acetylcholine receptors (nAChRs) play a pivotal role in synaptic transmission of neuronal signaling pathways and are fundamentally involved in neuronal disorders, including Alzheimer's disease, Parkinson's disease, and schizophrenia. In vertebrates, cholinergic pathways can be selectively inhibited by α-conotoxins; we show that in the model organism Drosophila, the cholinergic component of the giant fiber system is inhibited by α-conotoxins MII, AuIB, BuIA, EI, PeIA, and ImI. The injection of 45 pmol/fly of each toxin dramatically decreases the response of the giant fiber to dorsal longitudinal muscle (GF-DLM) connection to 20 ± 13.9% for MII; 26 ± 13.7% for AuIB, 12 ± 9.9% for BuIA, 30 ± 11.3% for EI, 1 ± 1% for PeIA, and 34 ± 15.4% for ImI. Through bioassay-guided fractionation of the venom of Conus brunneus, we found BruIB, an α-conotoxin that inhibits Drosophila nicotinic receptors but not its vertebrate counterparts. GF-DLM responses decreased to 43.7 ± 8.02% on injection of 45 pmol/fly of BruIB. We manipulated the Dα7 nAChR to mimic the selectivity of its vertebrate counterpart by placing structurally guided point mutations in the conotoxin-binding site. This manipulation rendered vertebrate-like behavior in the Drosophila system, enhancing the suitability of Drosophila as an in vivo tool to carry out studies related to human neuronal diseases. .
Project description:Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. alpha-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of alpha-conotoxins may serve as a platform for development of new therapies. Previously characterized alpha-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel alpha-conotoxin LtIA, which lacks this highly conserved motif but potently blocked alpha3beta2 nAChRs with a 9.8 nm IC(50) value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-block of alpha3beta2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR beta subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for alpha3beta2 versus alpha3beta4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC(50) value on alpha3beta2(F119Q) versus wild type nAChRs, whereas T59K and V111I beta2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha3beta2 nAChR that includes beta2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.
Project description:?7 nicotinic acetylcholine receptors (nAChRs) are ubiquitous in the nervous system and ensure important neurophysiological functionality for many processes. However, they are also found in cells of the immune system, where their role has been less studied. Here we report the pro-inflammatory effect of ImI, a well characterized conotoxin that inhibits ?7 nAChRs, on differentiated THP-1 pre-monocyte macrophages (MDM) obtained by phorbol 12-myristate 13 acetate (PMA) treatment. Enzyme-linked immunosorbent assay (ELISA) performed on supernatant fluids of LPS challenged MDM showed ImI-mediated upregulation of pro-inflammatory cytokine TNF-? in an ImI concentration-dependent manner from 0.5 to 5.0 µmol/L and for IL-8 up to 1.0 µmol/L. Levels of anti-inflammatory cytokine TGF-? remained practically unaffected in ImI treated MDMs. Nicotine at 10 µmol/L significantly downregulated the release of TNF-?, but showed a lesser effect on IL-8 secretion and no effect on TGF-?. Fluorescent competitive assays involving ImI, ?-bungarotoxin and nicotine using MDM and the murine macrophage RAW 264.7 suggest a common binding site in the ?7 receptor. This work extends the application of conotoxins as molecular probes to non-excitatory cells, such as macrophages and supports the involvement of the ?7 nAChR in regulating the inflammatory response via the cholinergic anti-inflammatory pathway (CAP).
Project description:Acetylcholine binding proteins (AChBPs) are unique spatial homologs of the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), and they reproduce some pharmacological properties of nAChRs. X-ray crystal structures of A?hBP in complex with ?-conotoxins provide important insights into the interactions of ?-conotoxins with distinct nAChR subtypes. Although considerable efforts have been made to understand why ?-conotoxin GIC is strongly selective for ?3?2 nAChR, this question has not yet been solved. Here we present the structure of ?-conotoxin GIC in complex with Aplysia californica AChBP (Ac-AChBP) at a resolution of 2.1?Å. Based on this co-crystal structure complemented with molecular docking data, we suggest the key residues of GIC in determining its high affinity and selectivity for human ?3?2 vs ?3?4 nAChRs. These suggestions were checked by radioligand and electrophysiology experiments, which confirmed the functional role of detected contacts for GIC interactions with Ac-AChBP and ?3?2 nAChR subtypes. While GIC elements responsible for its high affinity binding with Ac-AChBP and ?3?2 nAChR were identified, our study also showed the limitations of computer modelling in extending the data from the X-ray structures of the AChBP complexes to all nAChR subtypes.
Project description:Nicotinic acetylcholine receptors (nAChR) are therapeutic targets for a range of human diseases. ?-Conotoxins are naturally occurring peptide antagonists of nAChRs that have been used as pharmacological probes and investigated as drug leads for nAChR related disorders. However, ?-conotoxin interactions have been mostly characterised at the ?7 and ?3?2 nAChRs, with interactions at other subtypes poorly understood. This study provides novel structural insights into the molecular basis for ?-conotoxin activity at ?3?4 nAChR, a therapeutic target where subtype specific antagonists have potential to treat nicotine addiction and lung cancer. A co-crystal structure of ?-conotoxin LsIA with Lymnaea stagnalis acetylcholine binding protein guided the design and functional characterisations of LsIA analogues that identified the minimum pharmacophore regulating ?3?4 antagonism. Interactions of the LsIA R10F with ?4 K57 and the conserved -NN- ?-conotoxin motif with ?4 I77 and I109 conferred ?3?4 activity to the otherwise inactive LsIA. Using these structural insights, we designed LsIA analogues with ?3?4 activity. This new understanding of the structural basis of protein-protein interactions between ?-conotoxins and ?3?4 may help rationally guide the development of ?3?4 selective antagonists with therapeutic potential.
Project description:alpha-Conotoxins are peptide neurotoxins isolated from venomous cone snails that display exquisite selectivity for different subtypes of nicotinic acetylcholine receptors (nAChR). They are valuable research tools that have profound implications in the discovery of new drugs for a myriad of neuropharmacological conditions. They are characterized by a conserved two-disulfide bond framework, which gives rise to two intervening loops of extensively mutated amino acids that determine their selectivity for different nAChR subtypes. We have used a multistep synthetic combinatorial approach using alpha-conotoxin ImI to develop potent and selective alpha(7) nAChR antagonists. A positional scan synthetic combinatorial library was constructed based on the three residues of the n-loop of alpha-conotoxin ImI to give a total of 10,648 possible combinations that were screened for functional activity in an alpha(7) nAChR Fluo-4/Ca2+ assay, allowing amino acids that confer antagonistic activity for this receptor to be identified. A second series of individual alpha-conotoxin analogs based on the combinations of defined active amino acid residues from positional scan synthetic combinatorial library screening data were synthesized. Several analogs exhibited significantly improved antagonist activity for the alpha(7) nAChR compared with WT-ImI. Binding interactions between the analogs and the alpha(7) nAChR were explored using a homology model of the amino-terminal domain based on a crystal structure of an acetylcholine-binding protein. Finally, a third series of refined analogs was synthesized based on modeling studies, which led to several analogs with refined pharmacological properties. Of the 96 individual alpha-conotoxin analogs synthesized, three displayed > or =10-fold increases in antagonist potency compared with WT-ImI.
Project description:Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that are involved in fast synaptic transmission and mediated physiological activities in the nervous system. ?-Conotoxin ImI exhibits subtype-specific blockade towards homomeric ?7 and ?9 receptors. In this study, we established a method to build a 2×ImI-dendrimer/h (human) ?7 nAChR model, and based on this model, we systematically investigated the molecular interactions between the 2×ImI-dendrimer and h?7 nAChR. Our results suggest that the 2×ImI-dendrimer possessed much stronger potency towards h?7 nAChR than the ?-ImI monomer and demonstrated that the linker between ?-ImI contributed to the potency of the 2×ImI-dendrimer by forming a stable hydrogen-bond network with h?7 nAChR. Overall, this study provides novel insights into the binding mechanism of ?-ImI dendrimer to h?7 nAChR, and the methodology reported here opens an avenue for the design of more selective dendrimers with potential usage as drug/gene carriers, macromolecular drugs, and molecular probes.