Muscarinic acetylcholine receptors are G protein-coupled receptors that respond to acetylcholine and play important signaling roles in the nervous system. There are five muscarinic receptor subtypes ...(M1R to M5R), which, despite sharing a high degree of sequence identity in the transmembrane region, couple to different heterotrimeric GTP-binding proteins (G proteins) to transmit signals. M1R, M3R, and M5R couple to the G
family, whereas M2R and M4R couple to the G
family. Here, we present and compare the cryo-electron microscopy structures of M1R in complex with G
and M2R in complex with G
The M1R-G
complex exhibits distinct features, including an extended transmembrane helix 5 and carboxyl-terminal receptor tail that interacts with G protein. Detailed analysis of these structures provides a framework for understanding the molecular determinants of G-protein coupling selectivity.
The development and validation of new peptide dihedral parameters are reported for the OPLS-AA force field. High accuracy quantum chemical methods were used to scan φ, ψ, χ1, and χ2 potential energy ...surfaces for blocked dipeptides. New Fourier coefficients for the dihedral angle terms of the OPLS-AA force field were fit to these surfaces, utilizing a Boltzmann-weighted error function and systematically examining the effects of weighting temperature. To prevent overfitting to the available data, a minimal number of new residue-specific and peptide-specific torsion terms were developed. Extensive experimental solution-phase and quantum chemical gas-phase benchmarks were used to assess the quality of the new parameters, named OPLS-AA/M, demonstrating significant improvement over previous OPLS-AA force fields. A Boltzmann weighting temperature of 2000 K was determined to be optimal for fitting the new Fourier coefficients for dihedral angle parameters. Conclusions are drawn from the results for best practices for developing new torsion parameters for protein force fields.
Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many ...neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions—both therapeutic and hallucinogenic—are not understood, although activation of the 5-HT2A serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH—a prototypical hallucinogen—in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.
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•Cryo-EM 5-HT2A serotonin receptor structure complexed with hallucinogen and Gαq•The hallucinogen 25CN-NBOH displaces “toggle switch” tryptophan•Interactions essential for Gαq-specific signaling identified•X-ray crystal structure of LSD complexed with 5-HT2A elucidated
Roth et al. reveal structurally how psychedelics, including LSD, psilocin, mescaline, and various N-BOH analogs, mediate their therapeutic and hallucinogenic effects by binding to and activating their molecular target, the serotonin (5-HT) 2A receptor coupled with G-protein Gαq.
Genetic influences on gene expression in the human fetal brain plausibly impact upon a variety of postnatal brain-related traits, including susceptibility to neuropsychiatric disorders. However, to ...date, there have been no studies that have mapped genome-wide expression quantitative trait loci (eQTL) specifically in the human prenatal brain.
We performed deep RNA sequencing and genome-wide genotyping on a unique collection of 120 human brains from the second trimester of gestation to provide the first eQTL dataset derived exclusively from the human fetal brain. We identify high confidence cis-acting eQTL at the individual transcript as well as whole gene level, including many mapping to a common inversion polymorphism on chromosome 17q21. Fetal brain eQTL are enriched among risk variants for postnatal conditions including attention deficit hyperactivity disorder, schizophrenia, and bipolar disorder. We further identify changes in gene expression within the prenatal brain that potentially mediate risk for neuropsychiatric traits, including increased expression of C4A in association with genetic risk for schizophrenia, increased expression of LRRC57 in association with genetic risk for bipolar disorder, and altered expression of multiple genes within the chromosome 17q21 inversion in association with variants influencing the personality trait of neuroticism.
We have mapped eQTL operating in the human fetal brain, providing evidence that these confer risk to certain neuropsychiatric disorders, and identifying gene expression changes that potentially mediate susceptibility to these conditions.
Drug discovery in the era of cryo-electron microscopy Robertson, Michael J.; Meyerowitz, Justin G.; Skiniotis, Georgios
Trends in biochemical sciences,
February 2022, 2022-02-00, 20220201, Letnik:
47, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Structure-based drug discovery (SBDD) is an indispensable approach for the design and optimization of new therapeutic agents. Here, we highlight the rapid progress that has turned cryo‐electron ...microscopy (cryoEM) into an exceptional SBDD tool, and the wealth of new structural information it is providing for high-value pharmacological targets. We review key advantages of a technique that directly images vitrified biomolecules without the need for crystallization; both in terms of a broader array of systems that can be studied and the different forms of information it can provide, including heterogeneity and dynamics. We discuss near- and far-future developments, working in concert towards achieving the resolution and throughput necessary for cryoEM to make a widespread impact on the SBDD pipeline.
CryoEM has recently obtained resolutions sufficient for informing SBDD.The use of cryoEM allows access to new types of structures for SBDD, such as transmembrane proteins and difficult-to-crystalize complexes.New computational tools combined with cryoEM data provide key data for understanding ligand–protein interactions.Advances in cryoEM are rapidly improving accessibility and throughput, including increased automation in both hardware and software, and technical advances to improve signal to noise ratios and speed.New computational tools are unharnessing the ability of cryoEM to capture intermediate and equilibrium states of ligand–protein complexes.
Circadian clocks are 24-h timing devices that phase cellular responses; coordinate growth, physiology, and metabolism; and anticipate the day-night cycle. Here we report sensitivity of the ...Arabidopsis thaliana circadian oscillator to sucrose, providing evidence that plant metabolism can regulate circadian function. We found that the Arabidopsis circadian system is particularly sensitive to sucrose in the dark. These data suggest that there is a feedback between the molecular components that comprise the circadian oscillator and plant metabolism, with the circadian clock both regulating and being regulated by metabolism. We used also simulations within a three-loop mathematical model of the Arabidopsis circadian oscillator to identify components of the circadian clock sensitive to sucrose. The mathematical studies identified GIGANTEA (GI) as being associated with sucrose sensing. Experimental validation of this prediction demonstrated that GI is required for the full response of the circadian clock to sucrose. We demonstrate that GI acts as part of the sucrose-signaling network and propose this role permits metabolic input into circadian timing in ARABIDOPSIS:
Cannabis elicits its mood-enhancing and analgesic effects through the cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR) that signals primarily through the adenylyl cyclase-inhibiting ...heterotrimeric G protein Gi. Activation of CB1-Gi signaling pathways holds potential for treating a number of neurological disorders and is thus crucial to understand the mechanism of Gi activation by CB1. Here, we present the structure of the CB1-Gi signaling complex bound to the highly potent agonist MDMB-Fubinaca (FUB), a recently emerged illicit synthetic cannabinoid infused in street drugs that have been associated with numerous overdoses and fatalities. The structure illustrates how FUB stabilizes the receptor in an active state to facilitate nucleotide exchange in Gi. The results compose the structural framework to explain CB1 activation by different classes of ligands and provide insights into the G protein coupling and selectivity mechanisms adopted by the receptor.
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•3 Å cryo-EM structure of the CB1-Gi complex bound to potent agonist MDMB-Fubinaca•MDMB-Fubinaca locks “toggle switch” residues F2003.36/W3566.48 in active conformation•Quantum mechanics calculations reveal the mechanism for the high affinity of Fubinaca•Molecular dynamic simulations reveal a path for ligand entry between TM1 and TM7
Looking at how a toxic, synthetic ligand locks cannabinoid receptor 1 into a signaling conformation points to ways to understand and modulate receptor activity.
Natural killer (NK) cells participate in innate and adaptive immuneresponses to obligate intracellular pathogens and malignant tumors. Twomajor NK cell subsets have been identified in humans:CD56dim ...CD16+ and CD56bright CD16−. RestingCD56dim CD16+ NK cells express CXCR1, CXCR2, CXCR3, CXCR4,and CX3CR1 but no detectable levels of CC chemokine receptors on thecell surface. They migrate vigorously in response to CXCL12 and CXC3L1.In contrast, resting CD56bright CD16− NK cells expresslittle CXCR1, CXCR2, and CXC3R1 but high levels of CCR5 and CCR7. Chemotaxis of CD56bright CD16− NK cells is stimulated mostpotently by CCL19, CCL21, CXCL10, CXCL11, and CXCL12. Followingactivation, NK cells can migrate in response to additional CC and CXCchemokines. Cytolytic activity of NK cells is augmented by CCL2, CCL3,CCL4, CCL5, CCL10, and CXC3L1. Moreover, proliferation of CD56dim CD16+ NK cells is costimulated by CCL19 and CCL21. Activated NK cells produce XCL1, CCL1, CCL3, CCL4, CCL5, CCL22, andCXCL8. Chemokines secreted by NK cells may recruit other effector cellsduring immune responses. Furthermore, CCL3, CCL4, and CCL5 produced byNK cells can inhibit in vitro replication of HIV. CCL3 and CXL10expression appear to be required for protective NK cell responses invivo to murine cytomegalovirus or Leishmania major,respectively. Moreover, NK cells participate in the in vivo rejectionof transduced tumor cells that produce CCL19 or CCL21. Thus, chemokinesappear to play an important role in afferent and efferent NK cellresponses to infected and neoplastic cells.
G protein-coupled receptors (GPCRs) and other membrane proteins are valuable drug targets, and their dynamic nature makes them attractive systems for study with molecular dynamics (MD) simulations ...and free energy approaches. Here, we report the development, implementation, and validation of OPLS-AA/M force field parameters to enable simulations of these systems. These efforts include the introduction of post-translational modifications including lipidations and phosphorylation. We also modify previously reported parameters for lipids to be more consistent with the OPLS-AA force field standard and extend their coverage. These new parameters are validated on a variety of test systems, with the results compared to high-level quantum mechanics calculations, experimental data, and simulations with other force fields. The results demonstrate that the new parameters reliably reproduce the behavior of membrane protein systems.
Circadian clocks provide a competitive advantage in an environment that is heavily influenced by the rotation of the Earth, by driving daily rhythms in behaviour, physiology and metabolism in ...bacteria, fungi, plants and animals. Circadian clocks comprise transcription-translation feedback loops, which are entrained by environmental signals such as light and temperature to adjust the phase of rhythms to match the local environment. The production of sugars by photosynthesis is a key metabolic output of the circadian clock in plants. Here we show that these rhythmic, endogenous sugar signals can entrain circadian rhythms in Arabidopsis thaliana by regulating the gene expression of circadian clock components early in the photoperiod, thus defining a 'metabolic dawn'. By inhibiting photosynthesis, we demonstrate that endogenous oscillations in sugar levels provide metabolic feedback to the circadian oscillator through the morning-expressed gene PSEUDO-RESPONSE REGULATOR 7 (PRR7), and we identify that prr7 mutants are insensitive to the effects of sucrose on the circadian period. Thus, photosynthesis has a marked effect on the entrainment and maintenance of robust circadian rhythms in A. thaliana, demonstrating that metabolism has a crucial role in regulation of the circadian clock.
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