Targeted Molecular Dynamics Study of C-Loop Closure and Channel Gating in Nicotinic Receptors

The initial coupling between ligand binding and channel gating in the human α7 nicotinic acetylcholine receptor (nAChR) has been investigated with targeted molecular dynamics (TMD) simulation. During the simulation, eight residues at the tip of the C-loop in two alternating subunits were forced to move toward a ligand-bound conformation as captured in the crystallographic structure of acetylcholine binding protein (AChBP) in complex with carbamoylcholine. Comparison of apo- and ligand-bound AChBP structures shows only minor rearrangements distal from the ligand-binding site. In contrast, comparison of apo and TMD simulation structures of the nAChR reveals significant changes toward the bottom of the ligand-binding domain. These structural rearrangements are subsequently translated to the pore domain, leading to a partly open channel within 4 ns of TMD simulation. Furthermore, we confirmed that two highly conserved residue pairs, one located near the ligand-binding pocket (Lys145 and Tyr188), and the other located toward the bottom of the ligand-binding domain (Arg206 and Glu45), are likely to play important roles in coupling agonist binding to channel gating. Overall, our simulations suggest that gating movements of the α7 receptor may involve relatively small structural changes within the ligand-binding domain, implying that the gating transition is energy-efficient and can be easily modulated by agonist binding/unbinding

Identification of a Negative Allosteric Site on Human α4β2 and α3β4 Neuronal Nicotinic Acetylcholine Receptors

Acetylcholine-based neurotransmission is regulated by cationic, ligand-gated ion channels called nicotinic acetylcholine receptors (nAChRs). These receptors have been linked to numerous neurological diseases and disorders such as Alzheimer's disease, Parkinson's disease, and nicotine addiction. Recently, a class of compounds has been discovered that antagonize nAChR function in an allosteric fashion. Models of human α4β2 and α3β4 nicotinic acetylcholine receptor (nAChR) extracellular domains have been developed to computationally explore the binding of these compounds, including the dynamics and free energy changes associated with ligand binding. Through a blind docking study to multiple receptor conformations, the models were used to determine a putative binding mode for the negative allosteric modulators. This mode, in close proximity to the agonist binding site, is presented in addition to a hypothetical mode of antagonism that involves obstruction of C loop closure. Molecular dynamics simulations and MM-PBSA free energy of binding calculations were used as computational validation of the predicted binding mode, while functional assays on wild-type and mutated receptors provided experimental support. Based on the proposed binding mode, two residues on the β2 subunit were independently mutated to the corresponding residues found on the β4 subunit. The T58K mutation resulted in an eight-fold decrease in the potency of KAB-18, a compound that exhibits preferential antagonism for human α4β2 over α3β4 nAChRs, while the F118L mutation resulted in a loss of inhibitory activity for KAB-18 at concentrations up to 100 µM. These results demonstrate the selectivity of KAB-18 for human α4β2 nAChRs and validate the methods used for identifying the nAChR modulator binding site. Exploitation of this site may lead to the development of more potent and subtype-selective nAChR antagonists which may be used in the treatment of a number of neurological diseases and disorders

GABA Binding to an Insect GABA Receptor: A Molecular Dynamics and Mutagenesis Study

RDL receptors are GABA-activated inhibitory Cys-loop receptors found throughout the insect CNS. They are a key target for insecticides. Here, we characterize the GABA binding site in RDL receptors using computational and electrophysiological techniques. A homology model of the extracellular domain of RDL was generated and GABA docked into the binding site. Molecular dynamics simulations predicted critical GABA binding interactions with aromatic residues F206, Y254, and Y109 and hydrophilic residues E204, S176, R111, R166, S176, and T251. These residues were mutated, expressed in Xenopus oocytes, and their functions assessed using electrophysiology. The data support the binding mechanism provided by the simulations, which predict that GABA forms many interactions with binding site residues, the most significant of which are cation-π interactions with F206 and Y254, H-bonds with E204, S205, R111, S176, T251, and ionic interactions with R111 and E204. These findings clarify the roles of a range of residues in binding GABA in the RDL receptor, and also show that molecular dynamics simulations are a useful tool to identify specific interactions in Cys-loop receptors

Blockade of Neuronal α7-nAChR by α-Conotoxin ImI Explained by Computational Scanning and Energy Calculations

α-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 α-conotoxin ImI to the α7-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 α-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 α-conotoxins

Analysis of structure and function of the serotonin type-3 receptor using site directed mutagenesis, structure activity relationship and chimeric constructs

Thesis (Ph.D.) University of Alaska Fairbanks, 2005The serotonin type-3 receptor (5-HT₃R) is a cation conducting ligand gated ion channel that mediates fast synaptic transmission. The 5-HT₃R belongs to the Cys loop superfamily of ligand gated ion channels that also includes the nicotinic acetylcholine, glycine and GABAa receptors. The 5-HT₃R has been implicated in several processes such as emesis, gastrointestinal motility, drug abuse, alcoholism and nociception. Studies involving the ligand-binding domain will thus aid in development of new drugs that modulate these physiological and pathophysiological processes. The ligand-binding site of this receptor is comprised of six putative loops, viz. loop A-F. The focus of this thesis was to study the interactions of both agonists and antagonists with the 5- HT₃R. Interactions of two agonists, 5-HT and mCPBG, with the loop C region of the receptor were studied employing biochemical and receptor modeling studies. These studies identify novel determinants of 5-HT and mCPBG interactions with the 5-HT3 receptor. Similar studies involving granisetron, a competitive 5-HT₃R antagonist also reveal novel amino acids that interact with this antagonist. In order to further understand antagonist interactions with this receptor, the approach of structure activity relationship (SAR) studies was also employed to study the functional group interactions of lerisetron, a novel 5-HT₃R antagonist. Taken together with data from loops A, B, D and E, these data reveal an emerging picture of ligand interactions with the 5-HT₃R

Conformational selection or induced fit? 50 years of debate resolved

Exactly 50 years ago, biochemists raised the question of the mechanism of the conformational change that mediates “allosteric” interactions between regulatory sites and biologically active sites in regulatory/receptor proteins. Do the different conformations involved already exist spontaneously in the absence of the regulatory ligands (Monod-Wyman-Changeux), such that the complementary protein conformation would be selected to mediate signal transduction, or do particular ligands induce the receptor to adopt the conformation best suited to them (Koshland-Nemethy-Filmer—induced fit)? This is not just a central question for biophysics, it also has enormous importance for drug design. Recent advances in techniques have allowed detailed experimental and theoretical comparisons with the formal models of both scenarios. Also, it has been shown that mutated receptors can adopt constitutively active confirmations in the absence of ligand. There have also been demonstrations that the atomic resolution structures of the same protein are essentially the same whether ligand is bound or not. These and other advances in past decades have produced a situation where the vast majority of the data using different categories of regulatory proteins (including regulatory enzymes, ligand-gated ion channels, G protein-coupled receptors, and nuclear receptors) support the conformational selection scheme of signal transduction

Molecular mechanisms of allosteric modulation of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) are part of the Cys-loop family of ligand-gated ion channels, and are implicated in a wide variety of neurological disorders such as nicotine addiction, schizophrenia, and cognitive dysfunction. Therefore, they represent a critical molecular target for drug development and targeted therapeutic intervention. Positive allosteric modulators (PAMs) of ligandgated ion channels have a unique therapeutic potential because they enhance synaptic transmission without disrupting the endogenous timing mechanisms. This research focused on the neuronal α7 nicotinic receptor because they are located both pre- and postsynaptically and can modulate glutamatergic and dopaminergic release in the brain regions involved in drug-seeking behaviors. Understanding the molecular mechanisms by which allosteric modulators enhance activation of neuronal nicotinic acetylcholine receptors is therefore critically important to the development of new drugs for research and therapeutics. Experiments with the Substituted Cysteine Accessibility Method indicate that two chemically different positive allosteric modulators, PNU-120596 and permeable divalent cations, cause structural transitions (or changes in local electrostatic potential) in the extracellular ligand binding domain of the α7 nicotinic receptor that are similar but not identical to those caused by the agonist, acetylcholine. These results suggest that positive allosteric modulators share a conserved mechanism to enhance receptor gating that is unrelated to the chemical structure of the molecule. As an additional approach to study gating of the nicotinic receptors, I developed homology models derived from the structures of bacterial Cys-loop receptors in the closed and open states. A comparison of electrophysiological MTSEA modification data against in silico calculations of solvent accessibility and electrostatic potential showed that electrostatic potential in the extracellular ligandbinding domain of the α7 nAChR is a better predictor of receptor gating from the closed to open states. Overall, this body of work has shown that positive allosteric modulators and agonists of the α7 nAChR induce similar conformational changes in the extracellularligand binding domain of the receptor by reducing the large electronegative potential energy along the ion-permeation pathway. A unifying model of receptor gating (electrostatic compensation) and future experiments designed to test this model are discussed