Methaqualone, a quinazolinone marketed commercially as Quaalude, is a central nervous system depressant that was used clinically as a sedative-hypnotic, then became a notorious recreational drug in the 1960s-80s. Due to its high abuse potential, medical use of methaqualone was eventually prohibited, yet it persists as a globally abused substance. Methaqualone principally targets GABAA receptors, which are the major inhibitory neurotransmitter-gated ion channels in the brain. The restricted status and limited accessibility of methaqualone have contributed to its pharmacology being understudied. Here, we use cryo-EM to localize the GABAA receptor binding sites of methaqualone and its more potent derivative, PPTQ, to the same intersubunit transmembrane sites targeted by the general anesthetics propofol and etomidate. Both methaqualone and PPTQ insert more deeply into subunit interfaces than the previously-characterized modulators. Binding of quinazolinones to this site results in widening of the extracellular half of the ion-conducting pore, following a trend among positive allosteric modulators in destabilizing the hydrophobic activation gate in the pore as a mechanism for receptor potentiation. These insights shed light on the underexplored pharmacology of quinazolinones and further elucidate the molecular mechanisms of allosteric GABAA receptor modulation through transmembrane binding sites.

The evolution of new traits enables expansion into new ecological and behavioural niches. Nonetheless, demonstrated connections between divergence in protein structure, function and lineage-specific behaviours remain rare. Here we show that both octopus and squid use cephalopod-specific chemotactile receptors (CRs) to sense their respective marine environments, but structural adaptations in these receptors support the sensation of specific molecules suited to distinct physiological roles. We find that squid express ancient CRs that more closely resemble related nicotinic acetylcholine receptors, whereas octopuses exhibit a more recent expansion in CRs consistent with their elaborated 'taste by touch' sensory system. Using a combination of genetic profiling, physiology and behavioural analyses, we identify the founding member of squid CRs that detects soluble bitter molecules that are relevant in ambush predation. We present the cryo-electron microscopy structure of a squid CR and compare this with octopus CRs1 and nicotinic receptors2. These analyses demonstrate an evolutionary transition from an ancestral aromatic 'cage' that coordinates soluble neurotransmitters or tastants to a more recent octopus CR hydrophobic binding pocket that traps insoluble molecules to mediate contact-dependent chemosensation. Thus, our study provides a foundation for understanding how adaptation of protein structure drives the diversification of organismal traits and behaviour.

Chemotactile receptors (CRs) are a cephalopod-specific innovation that allow octopuses to explore the seafloor via ‘taste by touch’ 1 . CRs diverged from nicotinic acetylcholine receptors to mediate contact-dependent chemosensation of insoluble molecules that do not readily diffuse in marine environments. Here we exploit octopus CRs to probe the structural basis of sensory receptor evolution. We present the cryo-electron microscopy structure of an octopus CR and compare it with nicotinic receptors to determine features that enable environmental sensation versus neurotransmission. Evolutionary, structural and biophysical analyses show that the channel architecture involved in cation permeation and signal transduction is conserved. By contrast, the orthosteric ligand-binding site is subject to diversifying selection, thereby mediating the detection of new molecules. Serendipitous findings in the cryo-electron microscopy structure reveal that the octopus CR ligand-binding pocket is exceptionally hydrophobic, enabling sensation of greasy compounds versus the small polar molecules detected by canonical neurotransmitter receptors. These discoveries provide a structural framework for understanding connections between evolutionary adaptations at the atomic level and the emergence of new organismal behaviour.

Natriuretic peptides (NPs) increase cGMP, show beneficial cardiovascular effects and regulate energy metabolism in other tissues. However, little is known about their direct effect on cardiac mitochondria and cardiomyocyte apoptosis. Here, we examined whether NPs increase cGMP around mitochondria and alter apoptosis in cardiomyocytes. We constructed a novel FRET-based biosensor with high selectivity towards cGMP and found that ANP and CNP increase cGMP at the outer mitochondrial membrane. Moreover, ANP and CNP increased phosphorylation of the pro-apoptotic protein Drp1 and CNP prevented fragmentation of mitochondria. Stimulating cardiomyocytes with ANP or CNP reduced apoptosis, caspase 9 activation and cytochrome c release, suggesting that NPs decrease apoptosis through the intrinsic pathway that involves mitochondria. We suggest that cGMP increase in the outer mitochondrial membrane microdomain that inhibits the pro-apoptotic protein Drp1, leading to reduced mitochondrial fragmentation and thereby reduced apoptosis.

Well-thought-out figures are essential to describe key findings in structural biology because the figures represent the actual experimental data. However, illustrating complicated 3D structural features as 2D images can be quite challenging; therefore, thorough structural analysis is needed to plan how to present the key structural features effectively. For a publication-quality figure, generating several images in different styles is highly recommended. Slight differences in viewpoint, angle, depth, lighting, and colors may significantly impact the final output. Different rendering methods (cartoon, cylinder, sphere, or stick) should also be seriously considered based on the purpose of the figures. User-friendly graphic software includes Chimera, ChimeraX, and PyMOL; these can analyze macromolecule structures and generate high-resolution images. Despite similar capabilities, they have pros and cons in terms of their functionality and user friendliness. Table 1Pros and cons of molecular visualization software Pros Cons Chimera • Easy handling and modification of cryogenic electron microscopy (cryo-EM) maps• Intuitive and user-friendly interface for beginners to advanced• Supports Python scripting• Well-documented user guide• Free of charge for academic users • Need to learn specific commands for advanced functions ChimeraX • Super user-friendly interface for beginners• Fetch AlphaFold predicted models directly with its error plot• Supports Python scripting• Well-documented user guide• Free of charge for academic users • Need to learn specific commands for advanced functions• Limited functionality, compared with Chimera PyMOL • Intuitive and user-friendly interface for all levels of users• Supports Python scripting• Well documented (PyMOLWiki) • Hard to handle and manipulate crystallography and cryo-EM maps• License needed for academic users Open table in a new tab

Cephalopods are well known for their intricate sensory organs, elaborate nervous systems, and sophisticated behaviors that are comparable to complex vertebrates, but with radically different organization. Thus, cephalopods provide striking examples of convergent and divergent evolution that can be leveraged to understand the molecular basis of novelty across levels of biological organization. However, molecular functions giving rise to novel behaviors among these fascinating animals remain relatively unexplored. Recent work revealed that chemosensory receptors in cephalopods are members of the Cys-loop receptor superfamily, which in mammals includes the nicotinic acetylcholine, 5-HT3, GABAA and glycine receptors. We sought to understand how this branch of a family best known for fast chemical neurotransmission evolved to sense signaling molecules in a marine environment. We first present the structure of an octopus chemotactile receptor, which is used by its arms for “taste by touch” exploration of the seafloor. Together with evolutionary, biophysical, and behavioral analyses, we show how strikingly novel structural adaptations facilitate the receptor's transition from an ancestral role in neurotransmission to a new function in contact-dependent chemosensation of greasy environmental agonists. We next focus on squid, a cephalopod with a wildly different predation strategy. We combine genetics, physiology, and behavior to discover a new class of ancient chemotactile receptor, obtain its structure with a novel agonist bound, and perform evolutionary analyses to relate adaptations in squid receptors to more elaborate expansions in octopus. We then place chemotactile and ancestral neurotransmitter receptors in a phylogenetically, structurally, and functionally defined evolutionary timeline to elucidate mechanisms of adaptive behavior.

Scientific figures not only complement articles but can enhance them. In this third TrendsTalk of the Special series: Scientific figure development, previous TIBS authors focus on different approaches to creating protein structure figures, considering questions such as: how do you decide how to represent your protein structure? What programs do you prefer and why? What aspects do you consider when generating a protein structure figure? Contributing to this article are Clemens Grimm, co-corresponding author of 'Cytoplasmic gene expression: lessons from poxviruses' [1] (Figure 2, for example); Bin Liu, corresponding author of 'Roles of zinc-binding domain of bacterial RNA polymerase in transcription' [2] (Figure 2 and others); Vanessa Flegler, first author of 'More than just closed and open: unraveling a mechanosensor' [3] (Figure 1); and Jeong Joo Kim, first author of 'Direct structural insights into GABAA receptor pharmacology' [4] (Figure 3)

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory (AI) pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here, we present a crystal structure of PKG Iβ in which the AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ AI sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.

GABAA receptors are pentameric ligand-gated ion channels that mediate most fast neuronal inhibition in the brain. In addition to their important physiological roles, they are noteworthy in their rich pharmacology; prominent drugs used for anxiety, insomnia, and general anesthesia act through positive modulation of GABAA receptors. Direct structural information for how these drugs work was absent until recently. Efforts in structural biology over the past few years have revealed how important drug classes and natural products interact with the GABAA receptor, providing a foundation for studies in dynamics and structure-guided drug design. Here, we review recent developments in GABAA receptor structural pharmacology, focusing on subunit assemblies of the receptor found at synapses.

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1–5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain. Cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics and benzodiazepines reveal both common and distinct transmembrane binding sites, and show that the mechanisms of action of anaesthetics partially overlap with those of benzodiazepines.