Chris Ulens

Achbp-Targeted Alpha-Conotoxin Correlates Distinct Binding Orientations with Nachr Subtype Selectivity

Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine‐binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand‐binding domain, to discover a novel α‐conotoxin (α‐TxIA) in the venom of Conus textile. α‐TxIA bound with high affinity to AChBPs from different species and selectively targeted the α3β2 nAChR subtype. A co‐crystal structure of Ac‐AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac‐AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co‐crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype‐selective ligands with therapeutic potential in nAChR‐related diseases.

Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines

GABAA receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABAA receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABAA receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.

Multisite Binding of a General Anesthetic to the Prokaryotic Pentameric Erwinia chrysanthemi Ligand-gated Ion Channel (ELIC)

Background: Pentameric ligand-gated ion channels are modulated by general anesthetics. Results: The crystal structure of ELIC in complex with bromoform reveals anesthetic binding in the channel pore and in novel sites in the transmembrane and extracellular domain. Conclusion: General anesthetics allosterically modulate channel function via multisite binding. Significance: Our data reveal detailed insight into multisite recognition of general anesthetics at the structural level. Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABAA/C receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7–β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels.

A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors.

Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic acetylcholine receptors (nAChR), respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT3R.

Structural insights into Cys-loop receptor function and ligand recognition

This review outlines recent insights into ligand recognition, channel gating and ion permeation for the family of pentameric ligand-gated ion channels (pLGICs). These receptors are involved in the fast inhibitory and excitatory neurotransmission. Prototypical anion-selective members are the γ-amino butyric acid type A (GABA(A)), γ-amino butyric acid type C (GABA(C)) and glycine receptor. The cation-selective members are the 5-HT3 serotonin and nicotinic acetylcholine (nACh) receptors. They are the target for a wide variety of drugs and dysfunction in these receptors is associated with several diseases. We summarize recent structural knowledge in combination with electrophysiological data and molecular dynamic simulations, thereby describing key features of ligand binding, channel gating and ion permeation. A conserved cation-π interaction between ligand and aromatic residues of the ligand binding site critically contributes to ligand recognition, as revealed by X-ray crystal structures of acetylcholine binding proteins, as well as the integral pLGICs, ELIC and GluCl. In addition, we summarize the possible downstream effects on gating of structural rearrangements in the extracellular ligand-binding domain, which mainly occur in loop C and loop F. These data are discussed in the context of different conformational states of the pore-forming domain observed in crystal structures of GLIC and GluCl, which likely represent the open pore conformation, and ELIC, which likely corresponds to a closed pore conformation. We conclude with a current structural view on the determinants of ion selection and permeation.

Use of Acetylcholine Binding Protein in the Search for Novel Alpha7 Nicotinic Receptor Ligands. In Silico Docking, Pharmacological Screening, and X-Ray Analysis.

Acetylcholine binding protein (AChBP) is widely considered as a functional and structural homologue of the ligand binding domain of Cys-loop receptors. We report the use of AChBP as template to identify ligands for the nicotinic receptors (nAChRs). An in silico screening protocol was set up and applied to crystal structures of AChBP. Several ligands containing a dibenzosuberyl moiety were identified and shown to bind with high affinity to AChBP and alpha7 nAChRs. Two high affinity ligands were cocrystallized with AChBP, revealing the binding mode in the orthosteric site. Functional studies revealed that these two ligands caused inhibition of the alpha7, alpha4beta2, and 5HT(3) receptors. The noncompetive blockade of the receptors suggests that these compounds act by steric hindrance of the channel. The analysis of the dual binding mode of these dibenzosuberyl-containing compounds can lead to better understanding of the complex mode of action of similar tricyclic ligands on Cys-loop receptors.

Molecular actions of smoking cessation drugs at α4β2 nicotinic receptors defined in crystal structures of a homologous binding protein

Partial agonists of the α4β2 nicotinic acetylcholine receptor (nAChR), such as varenicline, are therapeutically used in smoking cessation treatment. These drugs derive their therapeutic effect from fundamental molecular actions, which are to desensitize α4β2 nAChRs and induce channel opening with higher affinity, but lower efficacy than a full agonist at equal receptor occupancy. Here, we report X-ray crystal structures of a unique acetylcholine binding protein (AChBP) from the annelid Capitella teleta, Ct-AChBP, in complex with varenicline or lobeline, which are both partial agonists. These structures highlight the architecture for molecular recognition of these ligands, indicating the contact residues that potentially mediate their molecular actions in α4β2 nAChRs. We then used structure-guided mutagenesis and electrophysiological recordings to pinpoint crucial interactions of varenicline with residues on the complementary face of the binding site in α4β2 nAChRs. We observe that residues in loops D and E are molecular determinants of desensitization and channel opening with limited efficacy by the partial agonist varenicline. Together, this study analyzes molecular recognition of smoking cessation drugs by nAChRs in a structural context.

Two new scorpion toxins that target voltage-gated Ca2+ and Na+ channels.

This report describes the isolation, primary structure determination, and functional characterization of two similar toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively). KLII from P. granulatus is identical to kurtoxin from Parabuthus transvaalicus (a 63 amino-acid long toxin) whereas KLI is a new peptide containing 62 amino acid residues closely packed by four disulfide bridges with a molecular mass of 7244. Functional assays showed that both toxins, KLI and kurtoxin from P. granulatus, potently inhibit native voltage-gated T-type Ca(2+) channel activity in mouse male germ cells. In addition, KLI was shown to significantly affect the gating mechanisms of recombinant Na(+) channels and weakly block alpha(1)3.3Ca(V) channels expressed in Xenopus oocytes. KLI and kurtoxin from P. granulatus represent new probes to study the role of ion channels in germ cells, as well as in cardiac and neural tissue.

Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor

Significance In this study we take advantage of a recently described chimera of the α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine binding protein (AChBP), termed α7-AChBP. To date, more than 70 crystal structures have been determined for AChBP in complex with ligands that occupy the orthosteric binding site. Here, we use an innovative screening strategy to discover molecular fragments that occupy allosteric binding sites. In combination with X-ray crystallography we determine a molecular blueprint of three different allosteric sites in α7-AChBP. Using electrophysiological recordings on the human α7 nAChR we demonstrate that each of the three sites is involved in allosteric modulation of the receptor. Our study contributes to understanding the sites of allosteric binding in ion channels. The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential.

Structural Basis of Ligand Recognition in 5-Ht3 Receptors.

The 5‐HT3 receptor is a pentameric serotonin‐gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti‐emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5‐HT3 receptor. In the serotonin‐bound structure, we observe hydrophilic interactions with loop E‐binding site residues, which might enable transitions to channel opening. In the granisetron‐bound structure, we observe a critical cation–π interaction between the indazole moiety of the ligand and a cationic centre in loop D, which is uniquely present in the 5‐HT3 receptor. We use a series of chemically tuned granisetron analogues to demonstrate the energetic contribution of this electrostatic interaction to high‐affinity ligand binding in the human 5‐HT3 receptor. Our study offers the first structural perspective on recognition of serotonin and antagonism by anti‐emetics in the 5‐HT3 receptor.