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Transition towards an alternative intermediate conformation of apo A2aR in a DOPG membrane A) Comparison of the intermediate state crystal structure of A2aR (PDB entry: 2YDO, light green) with bound adenosine and an MD-generated apo conformation achieved within a DOPG membrane (orange, belonging to replica #1 from 1.6 μs), showing B) and C) selected residues delineating the orthosteric pocket. ECL2 and TM helices are labelled where applicable. D) Distance between TM3-TM7 (from Cα atoms of R1023.50 and Y2887.53, respectively) during MD simulations, starting from the inactive crystal structure (PDB entry: 4EIY). E) Distance between TM3-TM6 (from Cα atoms of R1023.50 and E2286.30, respectively). F) and G) Vertical movement of ECL2 and TM3, respectively. MD simulations are performed in quadruplicate. Corresponding flat-lines show the observed distance in the active (PDB entry: 6GDG) and inactive (PDB entry: 4EIY) A2aR crystal structures.
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The activation process of G protein-coupled receptors (GPCRs) has been extensively studied, both experimentally and computationally. In particular, Molecular Dynamics (MD) simulations have proven useful in exploring GPCR conformational space. The typical behaviour of class A GPCRs, when subjected to unbiased MD simulations from their crystallized i...
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Adenosine signaling through A2AR serves as a negative regulator of the immune system. Unique to this suppressive pathway is its ability to impact numerous stromal and immune cells. Additionally, tumors exhibit elevated concentrations of adenosine further advancing the pathway’s potential as a powerful target for activating anti-tumor immunity. The...
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... Notably, stimulation of A2AR expression on macrophages has been shown to release cholesterol, which inhibits the formation of foam cells (84). The structural dynamics of A2AR are influenced by its phospholipid environment and cholesterol, resulting in a propensity to bind lipid isoform modulators (87). In therapeutic applications, liposome treatments combined with adenosine or specific A2AR agonists have markedly improved joint scores in post-traumatic osteoarthritis rats and mice with high-fat diet-induced osteoarthritis (88). ...
Obstructive sleep apnea (OSA) is the most common type of sleep apnea, which leads to episodes of intermittent hypoxia due to obstruction of the upper airway. A key feature of OSA is the upregulation and stabilization of hypoxia-inducible factor 1 (HIF-1), a crucial metabolic regulator that facilitates rapid adaptation to changes in oxygen availability. Adenosine A2A receptor (A2AR), a major adenosine receptor, regulates HIF-1 under hypoxic conditions, exerting anti-inflammatory properties and affecting lipid metabolism. The present study explored the roles of A2AR in OSA regulation, specifically focusing on its effects via the PI3K/Akt/HIF-1 pathway. The findings enhance our understanding the pharmacological potential of A2AR in OSA management and suggest future research directions in exploring its clinical applications.
... The nature of surrounding lipids may also affect GPCR functions and oligomerization, either directly by specific interactions, or indirectly by modification of the environment fluidity [12,13]. For example, studies based on experimental approaches [14,15] or molecular dynamics (MD) simulations [16,17] report that anionic lipids maintain or favor the active state of different receptors. This may affect GPCR responses in various cardiovascular cell types such as cardiomyocytes, endothelium, and vascular smooth muscle cells, which contain anionic lipids [18][19][20][21]. ...
... The internal DOPG prevented any subsequent inward motion of TM7. Internalization of a DOPG molecule has previously been reported during a microsecond long classical MD simulation of A2aR in a DOPG bilayer [16]. ...
Environmental factors, including mechanical stress and surrounding lipids, can influence the response of GPCRs, such as the mechanosensitive angiotensin II type 1 receptor (AT1). To investigate the impact of these factors on AT1 activation, we developed a steered molecular dynamics simulations protocol based on quaternion formalism. In this protocol, a pulling force was applied to the N-terminus of transmembrane helix 6 (TM6) to induce the TM6 opening characteristic of activation. Subsequently, the simulations were continued without constraints to allow the receptor to relax around the novel TM6 conformation under different conditions. We analyzed the responses of AT1 to membrane stretching, modeled by applying surface tension, in different bilayers. In phosphocholine bilayers without surface tension, we could observe a transient atypical structure of AT1, with an outward TM7 conformation, at the beginning of the activation process. This atypical structure then evolved toward a pre-active structure with outward TM6 and inward TM7. Strikingly, the presence of anionic phosphoglycerol lipids and application of surface tension synergistically favored the atypical structure, which led to an increase in the cross-section area of the receptor intracellular domain. Lipid internalization and H-bonds between lipid heads and the receptor C-terminus increased in phosphoglycerol vs phosphocholine bilayers, but did not depend on surface tension. The difference in the cross-section area of the atypical and pre-active conformations makes the conformational transition sensitive to lateral pressure, and favors the atypical conformation upon surface tension. Anionic lipids act as allosteric modulators of the conformational transition, by stabilizing the atypical conformation. These findings contribute to decipher the mechanisms underlying AT1 activation, highlighting the influence of environmental factors on GPCR responses. Moreover, our results reveal the existence of intermediary conformations that depend on receptor environment and could be targeted in drug design efforts.
... We identified 4 positively charged residues predicted by the Orientation of Proteins in Membranes (OPM) database 35 to be near the lipid-bilayer boundary: R199 5.60 , H230 6.32 , K233 6.35 and R291 7.56 (superscripts denote the Ballesteros-Weinstein nomenclature 36 ) and in different conformations between agonistbound and antagonist-bound A 2A AR crystal structures (Fig. 4a). Though our experiments were carried out at neutral pH, H230 6.32 has been proposed to be positively charged 37 and was thus included in the current study. Positively charged amino acids are frequently found in position 6.32 in other GPCRs (see Discussion). ...
... Inspection of the structures representative of the most likely distance for each case reveals that when the H230 6.32 to R291 7.56 distance is shorter, reflecting an inactive-like state, H230 6.32 , K233 6.35 R291 7.56 form a tight cluster (far left panels, top and bottom). When the distance is longer, reflecting an active-like state, this cluster is split, and coordinates a glutamic acid from the α5 helix of the G-protein (top right panel), or a PS headgroup in the absence of G-protein (lower right panel). of β 2 AR 38 and A 2A AR 37 , active conformations of the receptors were proposed to be stabilized by increasing the receptor affinity for agonists and interactions with membrane-facing residues in TM6 and TM7. In contrast, experimentally we observed only relatively smaller differences in agonist binding among different lipid compositions, and we also propose that penetration of anionic lipids into the G protein binding site is important to stabilizing an active A 2A AR conformation (Fig. 5). ...
G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we explore the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids prime the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeds through a less favorable induced fit mechanism. In computational models, anionic lipids mimic interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly alters the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.
... We identified 4 positively charged residues predicted by the Orientation of Proteins in Membranes (OPM) database 35 to be near the lipid-bilayer boundary: R199 5.60 , H230 6.32 , K233 6.35 and R291 7.56 (superscripts denote the Ballesteros-Weinstein nomenclature 36 ) and in different conformations between agonist-bound and antagonist-bound A2AAR crystal structures (Fig. 4a). Though our experiments were carried out at neutral pH, H230 6.32 has been proposed to be positively charged 37 and was thus included in the current study. Positively charged amino acids are frequently found in position 6.32 in other GPCRs (see Discussion). ...
G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we visualized the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids primed the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeded through a less favorable induced fit mechanism. In computational models, anionic lipids mimicked interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly altered the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.
... Interestingly, in some cases, timescales possible to reach during all-atom unbiased MD with current hardware and software may allow for the observation of large-scale motions associated with allosteric modulation. For instance, Bruzzese et al. used unbiased MD with a long timestep of 4 fs to simulate adenosine A 2a receptor, preparing simulation boxes with two different agonists and two different membranes, composed of DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine) or DOPG (1,2dioleoyl-sn-glycero-3-phosphoglycerol) [82]. In this case, a modulatory effect of membrane lipids on receptor activation was observed. ...
Allosteric modulation of G protein-coupled receptors (GPCRs) is nowadays a hot topic in medicinal chemistry. Allosteric modulators, i.e., compounds which bind in a receptor site topologically distinct from orthosteric sites, exhibit a number of advantages. They are more selective, safer and display a ceiling effect which prevents overdosing. Allosteric modulators of dopamine D2 receptor are potential drugs against a number of psychiatric and neurological diseases, such as schizophrenia and Parkinson’s disease. In this review, an insightful summary of current research on D2 receptor modulators is presented, ranging from their pharmacology and structural aspects of ligand-receptor interactions to their synthesis.
... This further highlighted the importance of using physiological cholesterol concentration in MD simulations. Recently, Bruzzese et al. performed MD simulations to study the role of different lipids in activation of A 2A AR [131]. Inactive A 2A AR in the apo or agonist (ADO or NECA) bound conformations were used as starting structures in either 1,2-dioleoyl-sn-glycerol-3-phosphoglycerol (DOPG) or 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC) lipid bilayer. ...
G protein-coupled receptors (GPCRs) represent the largest family of human membrane proteins. Four subtypes of adenosine receptors (ARs), the A1AR, A2AAR, A2BAR and A3AR, each with a unique pharmacological profile and distribution within the tissues in the human body, mediate many physiological functions and serve as critical drug targets for treating numerous human diseases including cancer, neuropathic pain, cardiac ischemia, stroke and diabetes. The A1AR and A3AR preferentially couple to the Gi/o proteins, while the A2AAR and A2BAR prefer coupling to the Gs proteins. Adenosine receptors were the first subclass of GPCRs that had experimental structures determined in complex with distinct G proteins. Here, we will review recent studies in molecular simulations and computer-aided drug discovery of the adenosine receptors and also highlight their future research opportunities.
... This ionic lock allows the intracellular end of TM6 to be bent toward TM3 in the inactive conformation. 29,43 In the intermediate-active conformation, Arg102 of the E/ DRY motif no longer participates in the ionic lock but interacts with Tyr288 of the NPxxY motif, bringing TM3 closer to TM7. 44 TM6 is still held in place near TM7 by a network of hydrogen bonds involving residues on either side of the NPxxY motif, but it is no longer interacting with TM3 with the deflection of the ionic lock. ...
... 46 Since water molecules were removed, it would be relevant to evaluate their involvement in this interaction. 44 Moreover, after tilting, TM6 is maintained against TM5 by a salt bridge between Glu228 of the E/DRY pattern with Arg205. 45 It is also worth mentioning that other observed phenomena are not covered in depth in this paper. ...
As long as the structural study of molecular mechanisms requires multiple molecular dynamics reflecting contrasted bioactive states, the subsequent analysis of molecular interaction networks remains a bottleneck to be fairly treated and requires a user-friendly 3D view of key interactions. Structural Interaction Network Analysis Protocols (SINAPs) is a proprietary python tool developed to (i) quickly solve key interactions able to distinguish two protein states, either from two sets of molecular dynamics simulations or from two crystallographic structures, and (ii) render a user-friendly 3D view of these key interactions through a plugin of UCSF Chimera, one of the most popular open-source viewing software for biomolecular systems. Through two case studies, glucose transporter-1 (GLUT-1) and A2A adenosine receptor (A2AR), SINAPs easily pinpointed key interactions observed experimentally and relevant for their bioactivities. This very effective tool was thus applied to identify the amino acids involved in the molecular enzymatic mechanisms ruling the activation of an immunomodulator drug candidate, P28 glutathione-S-transferase (P28GST). SINAPs is freely available at https://github.com/ParImmune/SINAPs.
... Он играет важную роль во многих биологических функциях, таких как сердечный ритм и кровообращение, церебральный и почечный кровоток, иммунная функция, регуляция боли и сон. Он был вовлечен в патофизиологические состояния, такие как воспалительные заболевания и нейродегенеративные расстройства [17]. В исследовании Turčin A. и соавт. ...
Antipsychotic-induced parkinsonism is an undesirable reaction from the extrapyramidal system that occurs against the background of taking antipsychotics (AP), more often in patients with schizophrenia. Antipsychotic-induced parkinsonism belongs to the group of secondary parkinsonism. Its prevalence in the world is about 36%. It is assumed that this undesirable AP reaction is genetically determined. In recent years, numerous associative genetic studies of predisposition to the development of antipsychotic-induced parkinsonism have been conducted. However, the research results are contradictory. Purpose. Review of the results of studies of genetic predictors of antipsychotic-induced parkinsonism in patients with schizophrenia. Materials and methods. We searched for full-text publications in Russian and English in the RSCI, PubMed, Web of Science, Springer databases using keywords and combined searches for words over the past decade. Results. The review considers candidate genes encoding proteins/enzymes involved in the pharmacodynamics and pharmacokinetics of AP. We analyzed 23 genome-wide studies examining 108 genetic variations, including SNV/polymorphisms of 26 candidate genes involved in the development of AIP in schizophrenic patients. Among such a set of obtained results, only 22 positive associations were revealed: rs1799732 (141CIns/Del), rs1800497 (C/T), rs6275 (C/T) DRD2; rs167771 (G/A) DRD3; VNTR*9R DAT1; rs4680 (G/A) СOMT; rs6311 (C/T) 5HTR2A; rs6318 (C/G), rs3813929 (С/Т), haplotype-997G, -759C, -697C и 68G HTR2C; rs2179652 (C/T), rs2746073 (T/A), rs4606 (C/G), rs1152746 (A/G), rs1819741 (С/Т), rs1933695 (G/A), haplotype rs1933695-G, rs2179652-C, rs4606-C, rs1819741-T и rs1152746-G, haplotype rs1933695-G, rs2179652-T, rs4606-G, rs1819741-C и rs1152746-A RGS2; haplotype TCCTC ADORA2A; rs4795390 (C/G) PPP1R1B; rs6265 (G/A) BDNF; rs12678719 (C/G) ZFPM2; rs938112 (C/A) LSMAP; rs2987902 (A/T) ABL1; HLA-B44; rs16947 (A/G), rs1135824 (A/G), rs3892097 (A/G), rs28371733 (A/G), rs5030867 (A/C), rs5030865 (A/C), rs1065852 (C/T), rs5030863 (C/G), rs5030862 (A/G), rs28371706 (C/T), rs28371725 (A/G), rs1080983 (A/G) CYP2D6. However, at the present time it should be recognized that there is no final or unique decision about the leading role of any particular SNV/polymorphism in the development of AIP. Conclusion. Disclosure of genetic predictors of AP-induced parkinsonism development may provide a key to the development of a strategy for personalized prevention and treatment of the neurological complication of AP-therapy of schizophrenia in real clinical practice.
... Negatively charged lipids also influence the activation state of the receptor (Dawaliby et al., 2016) and recent MD simulations have suggested two possible underlying mechanisms. Firstly, negatively charged lipids can intercalate between transmembrane helices (TM) 6 and 7 and block the ionic lock interaction that stabilizes the inactive state of the receptor (Neale et al., 2015;Bruzzese et al., 2020). Alternatively, attachment of ICL3 to a negatively charged membrane surface was suggested to contribute to the stabilization of the outward tilt of TM6 in the active state of the receptor (Bruzzese et al., 2018;Díaz et al., 2019). ...
... The comparison of membrane binding probabilities obtained in free and REST MD simulations is shown in Supplementary Figure S1. Figure 1A, top shows that the nonphosphorylated ICL3 of the β 2 AR in complex with the agonist adrenaline (β 2 AR*) binds to the negatively charged membrane composed of negatively charged 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (36:54:10 molar ratio). This finding agrees with previous MD simulations of the β 2 AR, adenosine A1 receptor, and cannabinoid receptor type 1 in single-component lipid membranes (Bruzzese et al., 2018;Díaz et al., 2019;Bruzzese et al., 2020). Interestingly, only the TM6-proximal half of ICL3 is membrane attached. ...
G protein-coupled receptors (GPCRs) are the largest class of human membrane proteins that bind extracellular ligands at their orthosteric binding pocket to transmit signals to the cell interior. Ligand binding evokes conformational changes in GPCRs that trigger the binding of intracellular interaction partners (G proteins, G protein kinases, and arrestins), which initiate diverse cellular responses. It has become increasingly evident that the preference of a GPCR for a certain intracellular interaction partner is modulated by a diverse range of factors, e.g., ligands or lipids embedding the transmembrane receptor. Here, by means of molecular dynamics simulations of the β 2 -adrenergic receptor and β-arrestin2, we study how membrane lipids and receptor phosphorylation regulate GPCR-arrestin complex conformation and dynamics. We find that phosphorylation drives the receptor’s intracellular loop 3 (ICL3) away from a native negatively charged membrane surface to interact with arrestin. If the receptor is embedded in a neutral membrane, the phosphorylated ICL3 attaches to the membrane surface, which widely opens the receptor core. This opening, which is similar to the opening in the G protein-bound state, weakens the binding of arrestin. The loss of binding specificity is manifested by shallower arrestin insertion into the receptor core and higher dynamics of the receptor-arrestin complex. Our results show that receptor phosphorylation and the local membrane composition cooperatively fine-tune GPCR-mediated signal transduction. Moreover, the results suggest that deeper understanding of complex GPCR regulation mechanisms is necessary to discover novel pathways of pharmacological intervention.
... Experimental studies have also been carried out that provide evidence of the activation of transmembrane proteins as a result of alterations to the lipid composition, e.g., using the mild detergent Triton X-100 [541,542], the relationship between lateral pressure profile and lipid composition was investigated both experimentally [697] and through MD simulation [698]. A few MD simulation studies found specific effects due to direct lipid interaction on protein behavior e.g., [699][700][701][702][703][704][705]; in most cases, the cholesterol molecules in the membrane were, however, most frequently shown to play the role of modulating the behavior of membrane proteins [706][707][708][709][710]. ...
We review the use of molecular dynamics (MD) simulation as a drug design tool in the context of the role that the lipid membrane can play in drug action, i.e., the interaction between candidate drug molecules and lipid membranes. In the standard “lock and key” paradigm, only the interaction between the drug and a specific active site of a specific protein is considered; the environment in which the drug acts is, from a biophysical perspective, far more complex than this. The possible mechanisms though which a drug can be designed to tinker with physiological processes are significantly broader than merely fitting to a single active site of a single protein. In this paper, we focus on the role of the lipid membrane, arguably the most important element outside the proteins themselves, as a case study. We discuss work that has been carried out, using MD simulation, concerning the transfection of drugs through membranes that act as biological barriers in the path of the drugs, the behavior of drug molecules within membranes, how their collective behavior can affect the structure and properties of the membrane and, finally, the role lipid membranes, to which the vast majority of drug target proteins are associated, can play in mediating the interaction between drug and target protein. This review paper is the second in a two-part series covering MD simulation as a tool in pharmaceutical research; both are designed as pedagogical review papers aimed at both pharmaceutical scientists interested in exploring how the tool of MD simulation can be applied to their research and computational scientists interested in exploring the possibility of a pharmaceutical context for their research.
