Structure of tetrameric forms of the serotonin-gated 5-HT3 receptor ion channel.

Multimeric membrane proteins are produced in the endoplasmic reticulum and transported to their target membranes which, for ion channels, is typically the plasma membrane. Despite the availability of many fully assembled channel structures, our understanding of assembly intermediates, multimer assembly mechanisms, and potential functions of non-standard assemblies is limited. We demonstrate that the pentameric ligand-gated serotonin 5-HT3A receptor (5-HT3AR) can assemble to tetrameric forms and report the structures of the tetramers in plasma membranes of cell-derived microvesicles and in membrane memetics using cryo-electron microscopy and tomography.

Computational drug development for membrane protein targets.

The application of computational biology in drug development for membrane protein targets has experienced a boost from recent developments in deep learning-driven structure prediction, increased speed and resolution of structure elucidation, machine learning structure-based design and the evaluation of big data. Recent protein structure predictions based on machine learning tools have delivered surprisingly reliable results for water-soluble and membrane proteins but have limitations for development of drugs that target membrane proteins. Structural transitions of membrane proteins have a central role during transmembrane signaling and are often influenced by therapeutic compounds.

Cryo-EM structures of ClC-2 chloride channel reveal the blocking mechanism of its specific inhibitor AK-42.

ClC-2 transports chloride ions across plasma membranes and plays critical roles in cellular homeostasis. Its dysfunction is involved in diseases including leukodystrophy and primary aldosteronism. AK-42 was recently reported as a specific inhibitor of ClC-2.

Applications and prospects of cryo-EM in drug discovery.

Drug discovery is a crucial part of human healthcare and has dramatically benefited human lifespan and life quality in recent centuries, however, it is usually time- and effort-consuming. Structural biology has been demonstrated as a powerful tool to accelerate drug development. Among different techniques, cryo-electron microscopy (cryo-EM) is emerging as the mainstream of structure determination of biomacromolecules in the past decade and has received increasing attention from the pharmaceutical industry.

Cryo-EM structure of human heptameric pannexin 2 channel.

Pannexin 2 (Panx2) is a large-pore ATP-permeable channel with critical roles in various physiological processes, such as the inflammatory response, energy production and apoptosis. Its dysfunction is related to numerous pathological conditions including ischemic brain injury, glioma and glioblastoma multiforme. However, the working mechanism of Panx2 remains unclear.

Molecular basis of ligand selectivity for melatonin receptors.

Sleep disorders in adults are related to adverse health effects such as reduced quality of life and increased mortality. About 30-40% of adults are suffering from different sleep disorders. The human melatonin receptors (MT1 and MT2) are family A G protein-coupled receptors that respond to the neurohormone melatonin MEL which regulates circadian rhythm and sleep.

Asymmetric opening of the homopentameric 5-HT serotonin receptor in lipid bilayers.

Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop receptor family are key players in fast signal transduction throughout the nervous system. They have been shown to be modulated by the lipid environment, however the underlying mechanism is not well understood. We report three structures of the Cys-loop 5-HT serotonin receptor (5HTR) reconstituted into saposin-based lipid bilayer discs: a symmetric and an asymmetric apo state, and an asymmetric agonist-bound state.

International Union of Basic and Clinical Pharmacology. CX. Classification of Receptors for 5-hydroxytryptamine; Pharmacology and Function.

5-HT receptors expressed throughout the human body are targets for established therapeutics and various drugs in development. Their diversity of structure and function reflects the important role 5-HT receptors play in physiologic and pathophysiological processes. The present review offers a framework for the official receptor nomenclature and a detailed understanding of each of the 14 5-HT receptor subtypes, their roles in the systems of the body, and, where appropriate, the (potential) utility of therapeutics targeting these receptors.

Enhancing the Signaling of GPCRs via Orthosteric Ions.

G protein-coupled receptors play essential roles in cellular processes such as neuronal signaling, vision, olfaction, tasting, and metabolism. As GPCRs are the most important drug targets, understanding their interactions with ligands is of utmost importance for discovering related new medicines. In many GPCRs, an allosteric sodium ion next to the highly conserved residue D has been proposed to stabilize the inactive receptor state by mediating interactions between transmembrane helices.

Advancing Drug Discovery via Artificial Intelligence.

Drug discovery and development are among the most important translational science activities that contribute to human health and wellbeing. However, the development of a new drug is a very complex, expensive, and long process which typically costs 2.6 billion USD and takes 12 years on average.

Computational modeling of the olfactory receptor Olfr73 suggests a molecular basis for low potency of olfactory receptor-activating compounds.

The mammalian olfactory system uses hundreds of specialized G-protein-coupled olfactory receptors (ORs) to discriminate a nearly unlimited number of odorants. Cognate agonists of most ORs have not yet been identified and potential non-olfactory processes mediated by ORs are unknown. Here, we used molecular modeling, fingerprint interaction analysis and molecular dynamics simulations to show that the binding pocket of the prototypical olfactory receptor Olfr73 is smaller, but more flexible, than binding pockets of typical non-olfactory G-protein-coupled receptors.

New Binding Sites, New Opportunities for GPCR Drug Discovery.

Many central biological events rely on protein-ligand interactions. The identification and characterization of protein-binding sites for ligands are crucial for the understanding of functions of both endogenous ligands and synthetic drug molecules. G protein-coupled receptors (GPCRs) typically detect extracellular signal molecules on the cell surface and transfer these chemical signals across the membrane, inducing downstream cellular responses via G proteins or β-arrestin.

Exploring a new ligand binding site of G protein-coupled receptors.

Identifying a target ligand binding site is an important step for structure-based rational drug design as shown here for G protein-coupled receptors (GPCRs), which are among the most popular drug targets. We applied long-time scale molecular dynamics simulations, coupled with mutagenesis studies, to two prototypical GPCRs, the M3 and M4 muscarinic acetylcholine receptors. Our results indicate that unlike synthetic antagonists, which bind to the classic orthosteric site, the endogenous agonist acetylcholine is able to diffuse into a much deeper binding pocket.

Single-Vesicle Assays Using Liposomes and Cell-Derived Vesicles: From Modeling Complex Membrane Processes to Synthetic Biology and Biomedical Applications.

The plasma membrane is of central importance for defining the closed volume of cells in contradistinction to the extracellular environment. The plasma membrane not only serves as a boundary, but it also mediates the exchange of physical and chemical information between the cell and its environment in order to maintain intra- and intercellular functions. Artificial lipid- and cell-derived membrane vesicles have been used as closed-volume containers, representing the simplest cell model systems to study transmembrane processes and intracellular biochemistry.

Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor.

There is growing interest in the use of mammalian protein expression systems, and in the use of antibody-derived chaperones, for structural studies. Here, we describe protocols ranging from the production of recombinant membrane proteins in stable inducible cell lines to biophysical characterization of purified membrane proteins in complex with llama antibody domains. These protocols were used to solve the structure of the mouse 5-HT3 serotonin receptor but are of broad applicability for crystallization or cryo-electron microscopy projects.

Two-dimensional crystallization of the mouse serotonin 5-HT receptor.

The mouse serotonin 5-HT receptor is a homo-pentameric ligand-gated ion channel (pLGIC) mediating fast excitatory neurotransmission in the central nervous system. The molecular mechanism of ion permeation of 5-HT receptors triggered by the neurotransmitter serotonin is not yet fully understood. The recent X-ray structure of the mouse serotonin 5-HT receptor in complex with a stabilizing nanobody revealed for the first time the entire structure of a mammalian pLGIC in detergent.

The Molecular Mechanism of P2Y1 Receptor Activation.

Human purinergic G protein-coupled receptor P2Y1 (P2Y1 R) is activated by adenosine 5'-diphosphate (ADP) to induce platelet activation and thereby serves as an important antithrombotic drug target. Crystal structures of P2Y1 R revealed that one ligand (MRS2500) binds to the extracellular vestibule of this GPCR, whereas another (BPTU) occupies the surface between transmembrane (TM) helices TM2 and TM3. We introduced a total of 20 μs all-atom long-timescale molecular dynamic (MD) simulations to inquire why two molecules in completely different locations both serve as antagonists while ADP activates the receptor.

Mechanistic Studies on the Stereoselectivity of the Serotonin 5-HT1A Receptor.

G-protein-coupled receptors (GPCRs) are involved in a wide range of physiological processes, and they have attracted considerable attention as important targets for developing new medicines. A central and largely unresolved question in drug discovery, which is especially relevant to GPCRs, concerns ligand selectivity: Why do certain molecules act as activators (agonists) whereas others, with nearly identical structures, act as blockers (antagonists) of GPCRs? To address this question, we employed all-atom, long-timescale molecular dynamics simulations to investigate how two diastereomers (epimers) of dihydrofuroaporphine bind to the serotonin 5-HT1A receptor and exert opposite effects. By using molecular interaction fingerprints, we discovered that the agonist could mobilize nearby amino acid residues to act as molecular switches for the formation of a continuous water channel.

A Gating Mechanism of the Serotonin 5-HT3 Receptor.

Our recently solved high-resolution structure of the serotonin 5-HT3 receptor (5-HT3R) delivered the first detailed structural insights for a mammalian pentameric ligand-gated ion channel. Based on this structure, we here performed a total of 2.8-μs all-atom molecular dynamics simulations to unravel at atomic detail how neurotransmitter binding on the extracellular domain induces sequential conformational transitions in the receptor, opening an ion channel and translating a chemical signal into electrical impulses across the membrane.

NK Cells Respond to Haptens by the Activation of Calcium Permeable Plasma Membrane Channels.

Natural Killer (NK) cells mediate innate immunity to infected and transformed cells. Yet, NK cells can also mount hapten-specific recall responses thereby contributing to contact hypersensitivity (CHS). However, since NK cells lack antigen receptors that are used by the adaptive immune system to recognize haptens, it is not clear if NK cells respond directly to haptens and, if so, what mediates these responses.