G protein-coupled receptors (GPCRs) are the largest superfamily of membrane receptors. They regulate physiological and pathological processes such as metabolic homeostasis, cell proliferation and differentiation, and the immune response, and are one of the most important classes of drug targets, being targeted by 30-40 % of marketed drugs. A growing number of studies continue to reveal the complexity of GPCRs, especially their ability to interact with each other to form higher-order structures such as homodimers and heterodimers, which have different functions than monomers, and are involved in disease development and progression. The existence of GPCR homodimers and heterodimers is opening up new directions in drug discovery and development to harness their therapeutic potential. Particularly striking is the ability of GPCR dimers to trigger unique biased signalling pathways, which exquisitely balance the relationship between therapeutic effects and side effects. By suppressing adverse reactions and enhancing beneficial drug effects, GPCR dimers provide an unprecedented opportunity to minimise side effects, maximise therapeutic efficacy and enhance safety. This review aims to highlight the latest research advances in GPCR dimerization and GPCR-biased signalling, focusing on the development of dimer-targeting and biased ligands as innovative drugs that will likely provide new strategies for treating GPCR-related diseases as well as a better understanding of drug design for compounds that target GPCRs. GPCRs will play an increasingly important role in precision medicine and personalised therapy, leading us towards a safer, more efficient and smarter pharmaceutical future.

Maintaining pH at cellular, tissular, and systemic levels is essential for human health. Proton-sensing GPCRs regulate physiological and pathological processes by sensing the extracellular acidity. However, the molecular mechanism of proton sensing and activation of these receptors remains elusive. Here, we present cryoelectron microscopy (cryo-EM) structures of human GPR4, a prototypical proton-sensing GPCR, in its inactive and active states. Our studies reveal that three extracellular histidine residues are crucial for proton sensing of human GPR4. The binding of protons induces substantial conformational changes in GPR4's ECLs, particularly in ECL2, which transforms from a helix-loop to a β-turn-β configuration. This transformation leads to the rearrangements of H-bond network and hydrophobic packing, relayed by non-canonical motifs to accommodate G proteins. Furthermore, the antagonist NE52-QQ57 hinders human GPR4 activation by preventing hydrophobic stacking rearrangement. Our findings provide a molecular framework for understanding the activation mechanism of a human proton-sensing GPCR, aiding future drug discovery.

The M2 muscarinic receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that serves as a model system for understanding ligand recognition and GPCR activation. Here, using vibrational spectroscopy, we identify the mechanisms governing M2R activation by its native agonist, acetylcholine. Combined with mutagenesis, computational chemistry, and organic synthetic chemistry, our analyses found that the precise distance between acetylcholine and Asn404, one of the amino acids constituting the ligand-binding site, is important for M2R activation and that the N404Q mutant undergoes partial active state-like conformational changes. We discovered that a water molecule bridging acetylcholine and Asn404 forms a precise and flexible hydrogen bond network, triggering the outward movement of transmembrane helix 6 in M2R. Consistent with this observation, disruptions in this hydrogen bond network via chemical modification at the α- or β-position of acetylcholine failed to activate M2R. Collectively, our findings pinpoint Asn404 as a critical residue that both senses acetylcholine binding and induces M2R activation.

Deep Mutational Scanning (DMS) is an emerging method to systematically test the functional consequences of thousands of sequence changes to a protein target in a single experiment. Because of its utility in interpreting both human variant effects and protein structure-function relationships, it holds substantial promise to improve drug discovery and clinical development. However, applications in this domain require improved experimental and analytical methods. To address this need, we report novel DMS methods to precisely and quantitatively interrogate disease-relevant mechanisms, protein-ligand interactions, and assess predicted response to drug treatment. Using these methods, we performed a DMS of the melanocortin-4 receptor (MC4R), a G-protein-coupled receptor (GPCR) implicated in obesity and an active target of drug development efforts. We assessed the effects of >6600 single amino acid substitutions on MC4R's function across 18 distinct experimental conditions, resulting in >20 million unique measurements. From this, we identified variants that have unique effects on MC4R-mediated Gαs- and Gαq-signaling pathways, which could be used to design drugs that selectively bias MC4R's activity. We also identified pathogenic variants that are likely amenable to a corrector therapy. Finally, we functionally characterized structural relationships that distinguish the binding of peptide versus small molecule ligands, which could guide compound optimization. Collectively, these results demonstrate that DMS is a powerful method to empower drug discovery and development.

The complement system plays crucial roles in innate immunity and inflammatory responses. The anaphylatoxin C3a mediates pro-inflammatory and chemotactic functions through the G protein-coupled receptor C3aR. While the active structure of the C3a-C3aR-Gi complex has been determined, the inactive conformation and activation mechanism of C3aR remain elusive. Here we report the cryo-EM structure of ligand-free, G protein-free C3aR, providing insights into its inactive conformation. In addition, we determine the structures of C3aR in complex with the synthetic small-molecule agonist JR14a in two distinct conformational states: a G protein-free intermediate, and a fully active Gi-bound state. The structure of the active JR14a-bound C3aR reveals that JR14a engages in highly conserved interactions with C3aR, similar to the binding of the C-terminal pentapeptide of C3a, along with JR14a-specific interactions. Structural comparison of C3aR in the apo, intermediate, and fully active states provides novel insights into the conformational landscape and activation mechanism of C3aR and defines a molecular basis explaining its high basal activity. Our results may aid in the rational design of therapeutics targeting complement-related inflammatory disorders.

GPR84 is an orphan GPCR that is expressed primarily in immune cells such as neutrophils and macrophages, and that modulates immune responses during inflammation. The receptor has appeared as a promising drug target, and accumulating evidence indicates that GPR84 inhibition is a viable approach for treatment of various inflammatory and fibrotic disorders. Herein, we report the discovery of a minor structural modification resulting in functional switch of agonists to inverse agonists. Subsequent SAR explorations led to the identification of low-nanomolar potency inverse agonists and antagonists, as exemplified by TUG-2181 (40g). Representative compounds exhibited good physicochemical properties, selectivity over other free fatty acid receptors, and the ability to fully inhibit GPR84-mediated neutrophil activation.

Understanding conformational changes in biomolecules is crucial for insights into their biological functions and drug design, often studied by molecular dynamics (MD) simulations. However, current measurements such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), interface area, and minimum distances fail to capture subtle conformational changes, including hydrophobic packing. Our study introduces gmx_RRCS, a precision tool developed to detect subtle conformational dynamics in MD simulations by analyzing residue-residue contact score (RRCS). This tool quantifies interaction strengths between residues, enabling systematic analysis of both major and subtle conformational changes. Its application in investigating the molecular recognition of peptide 20 by glucagon-like peptide-1 receptor (GLP-1R) quantified the interactions of specific peptide moieties with the receptor, identified crucial positions for receptor binding, and highlighted key receptor residues involved in peptide recognition throughout the MD simulation. In phosphoinositide 3-kinase alpha (PI3Kα), gmx_RRCS revealed distinct conformational states of oncogenic hotspot residues by quantifying subtle sidechain reorientations and salt bridge dynamics. Similarly, in nucleic acid systems, the tool distinguished differential binding mechanisms between ochratoxin A and norfloxacin by revealing unique interaction patterns at critical nucleobases correlating with binding affinities. The tool has been validated through the analysis of over 150 simulation trajectories, covering 40,000 ns of total simulation time and 20 systems. gmx_RRCS significantly advances structural/molecular biology studies by enhancing our understanding of protein conformational dynamics, thereby facilitating rational drug design. gmx_RRCS is freely available on GitHub (https://github.com/RuijinHospitalRCMSB/gmx_RRCS) and PyPI (https://pypi.org/project/gmx-RRCS).

G protein-coupled receptors (GPCRs) are the largest human membrane protein family that transduce extracellular signals into cellular responses. They are major pharmacological targets, with approximately 26% of marketed drugs targeting GPCRs, primarily at their orthosteric binding site. Despite their prominence, predicting the pharmacological effects of novel GPCR-targeting drugs remains challenging due to the complex functional dynamics of these receptors. Recent advances in X-ray crystallography, cryo-electron microscopy, spectroscopic techniques and molecular simulations have enhanced our understanding of receptor conformational dynamics and ligand interactions with GPCRs. These developments have revealed novel ligand-binding modes, mechanisms of action and druggable pockets. In this Review, we highlight such aspects for recently discovered small-molecule drugs and drug candidates targeting GPCRs, focusing on three categories: allosteric modulators, biased ligands, and bivalent and bitopic compounds. Although studies so far have largely been retrospective, integrating structural data on ligand-induced receptor functional dynamics into the drug discovery pipeline has the potential to guide the identification of drug candidates with specific abilities to modulate GPCR interactions with intracellular effector proteins such as G proteins and β-arrestins, enabling more tailored selectivity and efficacy profiles.

Different from reversible agonist-stimulated receptor activation, singlet oxygen oxidation activates permanently G protein-coupled receptor (GPCR) cholecystokinin 1 (CCK1R) in type II photodynamic action, with soluble photosensitizer dyes (sulphonated aluminum phthalocyanine, λmax 675 nm) or genetically encoded protein photosensitizers (KillerRed λmax 585 nm; mini singlet oxygen generator λmax 450 nm), together with a pulse of light (37 mW/cm2, 1-2 minutes). Three lines of evidence shed light on the mechanism of GPCR activated by singlet oxygen (GPCR-ABSO): (1) CCK1R is quantitatively converted from dimer to monomer; (2) Transmembrane domain 3, a pharmacophore for permanent photodynamic CCK1R activation, can be transplanted to non-susceptible M3 acetylcholine receptor; and (3) Larger size of disordered region in intracellular loop 3 correlates with higher sensitivity to photodynamic CCK1R activation. GPCR-ABSO will add to the arsenal of engineered designer GPCR such as receptors activated solely by synthetic ligands and designer receptors exclusively activated by designer drugs, but show some clear advantages: Enhanced selectivity (double selectivity of localized photosensitizer and light illumination), long-lasting activation with no need for repeated drug administration, antagonist-binding site remains intact when needed, ease to apply to multiple GPCR. This type of permanent photodynamic activation may be applied to functional proteins other than GPCR.

The free fatty acid receptor 2 (FFA2) is a G protein-coupled receptor (GPCR) that selectively recognizes short-chain fatty acids to regulate metabolic and immune functions. As a promising therapeutic target, FFA2 has been the focus of intensive development of synthetic ligands. However, the mechanisms by which endogenous and synthetic ligands modulate FFA2 activity remain unclear. Here, we present the structures of the human FFA2-Gi complex activated by the synthetic orthosteric agonist TUG-1375 and the positive allosteric modulator/allosteric agonist 4-CMTB, along with the structure of the inactive FFA2 bound to the antagonist GLPG0974. Structural comparisons with FFA1 and mutational studies reveal how FFA2 selects specific fatty acid chain lengths. Moreover, our structures reveal that GLPG0974 functions as an allosteric antagonist by binding adjacent to the orthosteric pocket to block agonist binding, whereas 4-CMTB binds the outer surface of transmembrane helices 6 and 7 to directly activate the receptor. Supported by computational and functional studies, these insights illuminate diverse mechanisms of ligand action, paving the way for precise GPCR-targeted drug design.

Calorie-rich foods, particularly those that are high in fat and sugar, evoke pleasure in both humans and animals1. However, prolonged consumption of such foods may reduce their hedonic value, potentially contributing to obesity2-4. Here we investigated this phenomenon in mice on a chronic high-fat diet (HFD). Although these mice preferred high-fat food over regular chow in their home cages, they showed reduced interest in calorie-rich foods in a no-effort setting. This paradoxical decrease in hedonic feeding has been reported previously3-7, but its neurobiological basis remains unclear. We found that in mice on regular diet, neurons in the lateral nucleus accumbens (NAcLat) projecting to the ventral tegmental area (VTA) encoded hedonic feeding behaviours. In HFD mice, this behaviour was reduced and uncoupled from neural activity. Optogenetic stimulation of the NAcLat→VTA pathway increased hedonic feeding in mice on regular diet but not in HFD mice, though this behaviour was restored when HFD mice returned to a regular diet. HFD mice exhibited reduced neurotensin expression and release in the NAcLat→VTA pathway. Furthermore, neurotensin knockout in the NAcLat and neurotensin receptor blockade in the VTA each abolished optogenetically induced hedonic feeding behaviour. Enhancing neurotensin signalling via overexpression normalized aspects of diet-induced obesity, including weight gain and hedonic feeding. Together, our findings identify a neural circuit mechanism that links the devaluation of hedonic foods with obesity.

The activation of G protein-coupled receptors (GPCRs) is a complex multibody multievent process involving agonist binding, receptor activation, G protein coupling, and subsequent G protein activation. The order and energetics of these events, though crucial for the rational design of selective GPCR drugs, are challenging to characterize and remain largely underexplored. Here, we employed molecular dynamics simulations and our recently developed traveling salesman-based automated path searching (TAPS) algorithm to efficiently locate the minimum free-energy paths for the coupling events of the CXC chemokine receptor 4 (CXCR4) with its endogenous ligand CXC chemokine ligand 12 (CXCL12) and Gi protein. We show that, after overcoming three low energy barriers (3.24-6.89 kcal/mol), Gi alone can precouple with CXCR4 even without CXCL12, consistent with previous reports on the existence of the apo CXCR4-Gi complex and our NanoBiT experiments. The highest barrier of 6.89 kcal/mol in this process corresponds to the packing of the proline-isoleucine-phenylalanine (PIF) motif of CXCR4. Interestingly, without Gi, CXCL12 alone cannot activate CXCR4 (high barrier of 18.89 kcal/mol). Instead, it can enhance Gi coupling by circumventing the energy barrier of PIF packing. Shedding new light on the activation mechanism of CXCR4, these results present TAPS as a promising tool for uncovering complete activation pathways of GPCRs and the corresponding agonist design.

CXCR4 (CXC chemokine receptor type 4), a member of the G protein-coupled receptor superfamily, plays a role in cell migration and functions as a coreceptor for HIV entry. Molecular therapeutics targeting CXCR4 have been under intensive investigation. To date, only two small-molecule antagonist drugs targeting CXCR4, plerixafor (AMD3100) and mavorixafor (AMD070), have been approved. Here, we present the high-resolution structures of CXCR4 complexed with AMD3100 and AMD070, as well as a small-molecule antagonist HF51116 that has very different chemical structure and binding mechanism from AMD3100 and AMD070. The interactions between these antagonists and the receptor are analyzed in details, and the mechanisms of antagonism are elucidated. Both the major and minor subpockets on CXCR4 are found to be involved in binding of these small-molecule antagonists. The distinct conformations of Trp942.60 observed in these structures highlight the plasticity of the binding pocket on CXCR4, offering valuable insights into the exploration and refinement of therapeutic strategies targeting this chemokine receptor.

Ran is a small GTPase of the Ras superfamily that governs nucleocytoplasmic transport, including that of miR-126, a microRNA that supports the homeostasis and expansion of leukemia stem cells (LSCs). Ran binds to Exportin 5 to facilitate the transport of precursor (pre)-miR-126 across the nuclear membrane for its maturation. Our goal is to inhibit Ran to prevent transport of pre-miR-126 to the cytoplasm. Like other Ras family proteins, targeting Ran with small molecules is challenging due to its relatively flat surface and lack of binding cavities. Ran's activity is regulated by a long and disordered C-terminus that provides opportunities for identifying cryptic binding pockets to target. We used a combination of molecular dynamics simulations and experiments and uncovered the critical role of the ensemble of the C-terminal conformations that enable the transition of Ran from the GTP-bound "on state" to its GDP-bound "off-state". We also showed that the Ran C-terminus allosterically modulates the conformations of residues in the nucleotide binding site and in the functionally relevant Switch 1 and 2 regions. Through computational deep mutational scans and experiments, we identified four residue hotspots L182, Y197, D200, and L201 at the core-C-terminus interface and four residue mutations V27A, E70D, N122A, and N122Y that mediate the allosteric communication between the core and switch regions. This information paves the way for our next step in the design of novel allosteric modulators for Ran.

Identifying target-specific drugs remains a challenge in pharmacology, especially for highly homologous proteins such as dopamine receptors D 2 R and D 3 R. Differences in target-specific cryptic druggable sites for such receptors arise from the distinct conformational ensembles underlying their dynamic behavior. While Molecular Dynamics (MD) simulations has emerged as a powerful tool for dissecting protein dynamics, the sheer volume of MD data requires scalable and unbiased data analysis strategies to pinpoint residue communities regulating conformational state ensembles. We have developed the Dynamically Resolved Universal Model for BayEsiAn network Tracking (DRUMBEAT) interpretable machine learning algorithm and validated it by identifying residue communities that enable the deactivation of the β 2 -adrenergic receptor. Further, upon analyzing dopamine receptor dynamics we identified distinct and non-conserved residue communities around the contacts F170 4.62 _F172 ECL2 and S146 4.38 _G141 34.56 that are specific to D 3 R conformational transitions compared to D 2 R. This information can be tapped to design subtype-specific drugs for neuropsychiatric and substance use disorders.

The increasing prevalence of overweight and obesity not only in adults but also among children and adolescents has become one of the most alarming health problems worldwide. Metabolic disorders accompanying fat accumulation during pathological weight gain induce chronic low-grade inflammation, which, in a vicious cycle, increases the immune response through pro-inflammatory changes in the cytokine (adipokine) profile. Obesity decreases life expectancy, largely because obese individuals are at an increased risk of many medical complications, often referred to as metabolic syndrome, which refers to the co-occurrence of insulin resistance (IR), impaired glucose tolerance, type 2 diabetes (T2D), atherogenic dyslipidemia, hypertension, and premature ischemic heart disease. Metabotropic G protein-coupled receptors (GPCRs) constitute the most numerous and diverse group of cell surface transmembrane receptors in eukaryotes. Among the GPCRs, researchers are focusing on the connection of G protein-coupled receptor 120 (GPR120), also known as free fatty acid receptor 4 (FFAR4), with signaling pathways regulating the inflammatory response and insulin sensitivity. This review presents the current state of knowledge concerning the involvement of GPR120 in anti-inflammatory and metabolic signaling. Since both inflammation in adipose tissue and insulin resistance are key problems in obesity, there is a rationale for the development of novel, GPR120-based therapies for overweight and obese individuals. The main problems associated with introducing this type of treatment into clinical practice are also discussed.

Current treatments of inflammatory bowel disease (IBD) largely depend on anti-inflammatory and immunosuppressive strategies with unacceptable efficacy and adverse events. Resolution or repair agents to treat IBD are not available but potential targets like formyl peptide receptor 2 (FPR2/ALX) may fill the gap. In this study we evaluated the therapeutic effects of two small molecule FPR2/ALX modulators (agonist Quin-C1 and antagonist Quin-C7) against IBD. We first analyzed the cryo-electron microscopy structure of the Quin-C1-FPR2 in complex with heterotrimeric Gi to reveal the structural basis for ligand recognition and FPR2 activation. We then established dextran sulfate sodium (DSS)-induced colitis model in both normal and myeloid depletion mice. We showed that oral administration of Quin-C1 for 7 days ameliorated DSS-induced colitis evidenced by alleviated disease activity indexes, reduced colonic histopathological scores, and corrected cytokine disorders. Meanwhile, we found that oral administration of FPR2/ALX antagonist Quin-C7 exerted therapeutic actions similar to those of Quin-C1. In terms of symptomatic improvements, the ED50 values of Quin-C1 and Quin-C7 were 1.3660 mg/kg and 2.2110 mg/kg, respectively. The underlying mechanisms involved ERK- or ERK/JNK-mediated myeloid cell regulation that limited the development of colitis and inflammation. This is the first demonstration of anti-colitis property caused by synthetic small molecule FPR2/ALX modulators, implying that FPR2/ALX modulation rather than agonism alone ameliorates IBD.

Despite the growing number of G protein-coupled receptor (GPCR) structures being resolved, the dynamic process of how GPCRs transit from the inactive toward the active state remains unclear. In this study, comprehensive molecular dynamics simulations were performed to explore how ligand binding modulates the conformational dynamics of the M2 muscarinic acetylcholine receptor (M2R). We observed a sequential occurrence of structural changes in the inactive-to-active transition of M2R induced by a superagonist iperoxo, which includes the orthosteric binding site contraction, the TM6 opening into an intermediate conformation, and a further structural change toward full activation upon binding to G protein or a G protein mimetic nanobody. Two activation intermediates were identified, which show structural features different from those reported for apo-GPCRs. Moreover, our results suggest that stabilization of a specific W4006.48 conformation and enhanced F3966.44 dynamics are crucial for activation, whereas distinct side-chain rotamer equilibriums of Y2065.58 in the cytoplasmic cavity are correlated with agonist efficacies. Our work provides atomic-level structural insights into the agonist-induced M2R activation pathway and highlights a mechanism by which ligand efficacy can be encoded and transduced in the form of aromatic ring dynamics.

Cytomegaloviruses (CMVs) encode viral G-protein-coupled receptors (vGPCRs) that have diverged from their cellular homologues to perform new functions. Human cytomegalovirus (HCMV) encodes four vGPCRs: UL33, UL78, US27, and US28, which contribute to viral pathogenesis, cellular signalling, and latency. While the role of US28 in chemokine signalling and viral latency is well characterised, the functions of other vGPCRs remain incompletely understood. Rodent cytomegaloviruses only have homologues to UL33 and UL78, while primates have two to five additional GPCRs which are homologues of US27 and US28. Different CMVs appear to have evolved vGPCRs with functions specific to infection of their respective host. As non-human CMVs are used as model organisms to understand clinical cytomegalovirus disease and develop vaccines and antivirals, understanding the differences between these vGPCRs helps researchers understand critical differences between their models. This review aims to address the differences between CMV vGPCRs, and how these differences may affect models of CMV disease to facilitate future research.

Sleep plays a role in the elimination of neurotoxic metabolites that are accumulated in the waking brain as a result of neuronal activity. Long-term insomnia and sleep deprivation are associated with oxidative stress, neuroinflammation, amyloid beta (Aβ) deposition, and Lewy body formation, which are known to increase the risk of mild cognitive impairment (MCI) and dementia. Orexin A (OXA) and orexin B (OXB), two neuropeptides produced in the lateral hypothalamus, are known to influence the sleep-wake cycle and the stress responses through their interactions with OX receptor 1 (OX1R) and OX receptor 2 (OX2R), respectively. OX/OXR cascade demonstrates intricate neuroprotective and anti-inflammatory effects by inhibiting nuclear factor-kappa B (NF-kB) and PLC/Ca2+ pathway activation. OX1R binds OXA more strongly than OXB by one-order ratio, whereas OX2R binds both OXA and OXB with equal strengths. Overexpression of OXs in individuals experiences sleep deprivation, circadian rhythm disturbances, insomnia-associated MCI, Parkinson's disease (PD), and Alzheimer's disease (AD). Many dual OXR antagonists (DORAs) have been effective in their clinical studies, with suvorexant and daridorexant receiving FDA clearance for insomnia therapy in 2014 and 2022 respectively. The results of clinical studies suggested that there is a new pharmaceutical option for treating insomnia and the sleep deprivation-AD/PD relationship by targeting the OXR system. DORAs treatment reduces Aβ deposition in the brain and improves synaptic plasticity and circadian expression. This review indicates the link between sleep disorders and MCI, DORAs are an appropriate medication category for treating insomnia, and sleep deprivation links AD and PD.

We present a series of three articles on the genetics and pharmacogenetics of G protein- coupled receptors (GPCR). In the first article, we discuss genetic variants of the G protein subunits and accessory proteins that are associated with human phenotypes; in the second article, we build upon this to discuss "G protein-coupled receptor (GPCR) gene variants and human genetic disease" and in the third article, we survey "G protein-coupled receptor pharmacogenomics". In the present article, we review the processes of ligand binding, GPCR activation, inactivation, and receptor trafficking to the membrane in the context of human genetic disease resulting from pathogenic variants of accessory proteins and G proteins. Pathogenic variants of the genes encoding G protein α and β subunits are examined in diverse phenotypes. Variants in the genes encoding accessory proteins that modify or organize G protein coupling have been associated with disease; these include the contribution of variants of the regulator of G protein signaling (RGS) to hypertension; the role of variants of activator of G protein signaling type III in phenotypes such as hypoxia; the contribution of variation at the RGS10 gene to short stature and immunological compromise; and the involvement of variants of G protein-coupled receptor kinases (GRKs), such as GRK4, in hypertension. Variation in genes that encode proteins involved in GPCR signaling are outlined in the context of the changes in structure and function that may be associated with human phenotypes.

Biased agonism in G protein-coupled receptors is a phenomenon resulting in the selective activation of distinct intracellular signaling pathways by different agonists, which may exhibit bias toward either Gs, Gi, or arrestin-mediated pathways. This study investigates the structural basis of ligand-induced biased agonism within the context of the β2-adrenergic receptor (β2-AR). Atomistic molecular dynamics simulations were conducted for β2-AR complexes with two stereoisomers of methoxynaphtyl fenoterol (MNFen), that is, compounds eliciting qualitatively different cellular responses. The simulations reveal distinct interaction patterns within the binding cavity, dependent on the stereoisomer. These changes propagate to the intracellular parts of the receptor, triggering various structural responses: the dynamic structure of the intracellular regions of the (R,R)-MNFen complex more closely resembles the "Gs-compatible" and "β-arrestin-compatible" conformation of β2-AR, while both stereoisomers maintain structural responses equidistant from the inactive conformation. These findings are confirmed by independent coarse-grained simulations. In the context of deciphered molecular mechanisms, Trp313 plays a pivotal role, altering its orientation upon interactions with (R,R)-MNFen, along with the Lys305-Asp192 ionic bridge. This effect, accompanied by ligand interactions with residues on TM2, increases the strength of interactions within the extracellular region and the binding cavity, resulting in a slightly more open conformation and a minor (by ca. 0.2 nm) increase in the distance between the TM5-TM7, TM1-TM6, TM6-TM7, and TM1-TM5 pairs. On the other hand, an even slighter decrease in the distance between the TM1-TM4 and TM2-TM4 pairs is observed.

Probing the differences between inactive/active states of the serotonin 1A receptor (5-HT1AR) and the dynamic receptor conformations is vital for understanding signaling transduction pathways and diverse physiological responses. Here, we compared the conformational features between the inactive and active states of 5-HT1AR and explored the role of serotonin in the activation process of 5-HT1AR by using molecular dynamics (MD) simulations. The results show that the position of TM6 and the arrangements of key motifs exhibit distinctions in the inactive and active states of 5-HT1AR. The binding of serotonin to 5-HT1AR is mostly driven by hydrophobic, aromatic stacking, anion-π, and H-bonding interactions. We also performed additional MD simulations with electric fields (EFs) of 0.01 and 0.03 V/nm to investigate the effects of EFs on the conformation of the 5-HT1AR-serotonin complex. The conformational change of 5-HT1AR and the inward movement of TM6 are increased with the field strength, indicative of a dependence on the strength of the EF. The EF of 0.03 V/nm affects the binding behaviors of serotonin with 5-HT1AR and further disturbs the activation of 5-HT1AR by serotonin. This study first reveals atomic-level information about the distinct features between inactive and active states of 5-HT1AR and demonstrates the pivotal role of EF in modulating the 5-HT1AR-ligand complex.

G protein-coupled sphingosine-1-phosphate receptor 1 (S1PR1), a drug target for inflammatory bowel disease (IBD), enables immune cells to egress from lymph nodes, but the treatment increases the risk of immunosuppression. The functional signaling pathway triggered by S1PR1 activation in endothelial cells and its therapeutic application remains unclear. Here, we showed that S1PR1 is highly expressed in endothelial cells of IBD patients and positively correlated with endothelial markers. Gi-biased agonist-SAR247799 activated S1PR1 and reversed pathology in male mouse and organoid IBD models by protecting the integrity of the endothelial barrier without affecting immune cell egress. Cryo-electron microscopy structure of S1PR1-Gi signaling complex bound to SAR247799 with a resolution of 3.47 Å revealed the recognition mode for the biased ligand. With the efficacy of SAR247799 in treating other endothelial dysfunction-associated inflammatory diseases, our study offers mechanistic insights into the Gi-biased S1PR1 agonist and represents a strategy for endothelial dysfunction-associated disease treatment.

Bayesian network modeling (BN modeling, or BNM) is an interpretable machine learning method for constructing probabilistic graphical models from the data. In recent years, it has been extensively applied to diverse types of biomedical data sets. Concurrently, our ability to perform long-time scale molecular dynamics (MD) simulations on proteins and other materials has increased exponentially. However, the analysis of MD simulation trajectories has not been data-driven but rather dependent on the user's prior knowledge of the systems, thus limiting the scope and utility of the MD simulations. Recently, we pioneered using BNM for analyzing the MD trajectories of protein complexes. The resulting BN models yield novel fully data-driven insights into the functional importance of the amino acid residues that modulate proteins' function. In this report, we describe the BaNDyT software package that implements the BNM specifically attuned to the MD simulation trajectories data. We believe that BaNDyT is the first software package to include specialized and advanced features for analyzing MD simulation trajectories using a probabilistic graphical network model. We describe here the software's uses, the methods associated with it, and a comprehensive Python interface to the underlying generalist BNM code. This provides a powerful and versatile mechanism for users to control the workflow. As an application example, we have utilized this methodology and associated software to study how membrane proteins, specifically the G protein-coupled receptors, selectively couple to G proteins. The software can be used for analyzing MD trajectories of any protein as well as polymeric materials.

PROteolysis TArgeting Chimeras (PROTACs), also known as ligand-directed degraders (LDDs), are an innovative class of small molecules that leverage the ubiquitin-proteasome system to induce the degradation of target proteins. Structure based design methods are not readily applicable for designing LDDs due to the dynamic nature of the ternary complexes. This study investigates the dynamic properties of five LDD-mediated BRD4-cereblon complexes, focusing on the challenges of evaluating linker efficiency due to the difficulty in identifying suitable computational metrics that correlate well with the cooperativity or degradation propensity of LDDs. We uncovered that protein frustration, a concept originally developed to understand protein folding, calculated for the residues in the protein-protein interface of the LDD-mediated ternary complexes recapitulate the strength of degradation of the LDDs. Our findings indicated that hydrophobic residues in the interface are among the highly frustrated residues pairs, and they are crucial in distinguishing strong degraders from weak ones. By analyzing frustration patterns, we identified key residues and interactions critical to the effectiveness of the ternary complex. These insights provide practical guidelines for designing and prioritizing more efficient degraders, paving the way for the development of next-generation LDDs with improved therapeutic potential.

Three proton-sensing G protein-coupled receptors (GPCRs)-GPR4, GPR65, and GPR68-respond to extracellular pH to regulate diverse physiology. How protons activate these receptors is poorly understood. We determined cryogenic-electron microscopy (cryo-EM) structures of each receptor to understand the spatial arrangement of proton-sensing residues. Using deep mutational scanning (DMS), we determined the functional importance of every residue in GPR68 activation by generating ∼9,500 mutants and measuring their effects on signaling and surface expression. Constant-pH molecular dynamics simulations provided insights into the conformational landscape and protonation patterns of key residues. This unbiased approach revealed that, unlike other proton-sensitive channels and receptors, no single site is critical for proton recognition. Instead, a network of titratable residues extends from the extracellular surface to the transmembrane region, converging on canonical motifs to activate proton-sensing GPCRs. Our approach integrating structure, simulations, and unbiased functional interrogation provides a framework for understanding GPCR signaling complexity.

Despite the widespread physiological roles of beta-arrestin (β-arr) coupling in G protein-coupled receptor (GPCR) regulation, the molecular basis of GPCR/β-arr interaction has been studied primarily in monomeric family A GPCRs. Here we take an integrative biophysical and structural approach to uncover extreme molecular diversity in β-arr coupling to the neuromodulatory metabotropic glutamate receptors (mGluRs), prototypical, dimeric family C GPCRs. Using a new single molecule pulldown assay, we find that mGluRs couple to β-arrs with a 2:1 or 2:2 stoichiometry via a combination of "tail" and "core" interactions. Single molecule FRET and electron microscopy show that β-arr1 stabilizes active conformations of mGluR8 and a combination of cryo-EM structures and molecular dynamics simulations define the positioning of mGluR8-bound β-arr1, together suggesting a steric mechanism of mGluR desensitization involving interactions with both subunits and the lipid bilayer. Finally, combinatorial mutagenesis enables the identification of a landscape of homo- and hetero-dimeric mGluR/β-arr complexes, including mGluR/β-arr1/β-arr2 megacomplexes, providing a framework for family C GPCR/β-arr coupling and expanding the known range of GPCR/transducer coupling modes.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in humans, playing a crucial role in regulating diverse cellular processes and serving as primary drug targets. Traditional drug design has primarily focused on ligands that uniformly activate or inhibit GPCRs. However, the concept of biased agonism-where ligands selectively stabilize distinct receptor conformations, leading to unique signaling outcomes-has introduced a paradigm shift in therapeutic development. Despite the promise of biased agonists to enhance drug efficacy and minimize side effects, a comprehensive understanding of the structural and biophysical mechanisms underlying biased signaling is essential. Recent advancements in GPCR structural biology have provided unprecedented insights into ligand binding, conformational dynamics, and the molecular basis of biased signaling. These insights, combined with improved techniques for characterizing ligand efficacy, have driven the development of biased ligands for several GPCRs, including opioid, angiotensin, and adrenergic receptors. This review synthesizes these developments, from mechanisms to drug discovery in biased signaling, emphasizing the role of structural insights in the rational design of next-generation biased agonists with superior therapeutic profiles. Ultimately, these advances hold the potential to revolutionize GPCR-targeted drug discovery, paving the way for more precise and effective treatments.

GPR20, an orphan G protein-coupled receptor (GPCR), shows significant expression in intestinal tissue and represents a potential therapeutic target to treat gastrointestinal stromal tumors. GPR20 performs high constitutive activity when coupling with Gi. Despite the pharmacological importance of GPCR constitutive activation, determining the mechanism has long remained unclear. In this study, we explored the constitutive activation mechanism of GPR20 through large-scale unbiased molecular dynamics simulations. Our results unveil the allosteric nature of constitutively activated GPCR signal transduction involving extracellular and intracellular domains. Moreover, the constitutively active state of the GPR20 requires both the N-terminal cap and Gi protein. The N-terminal cap of GPR20 functions like an agonist and mediates long-range activated conformational shift. Together with the previous study, this study enhances our knowledge of the self-activation mechanism of the orphan receptor, facilitates the drug discovery efforts that target GPR20.

Dopamine receptor D4 (DRD4) plays a vital role in regulating various physiological functions, including attention, impulse control, and sleep, as well as being associated with various neurological diseases, including attention deficit hyperactivity disorder, novelty seeking, and so on. However, a comprehensive analysis of harmful nonsynonymous single nucleotide polymorphisms (nsSNPs) of the DRD4 gene and their effects remains unexplored. The aim of this study is to uncover novel damaging missense nsSNPs and their structural and functional effects on the DRD4 receptor. From the dbSNP database, we found 677 nsSNPs, and then we analyzed their functional consequences, disease associations, and effects on protein stability with fifteen in silico tools. Five variants, including L65ICL1P (rs1459150721), V1163.33D (rs761875546), I1293.46S (rs751467198), I1564.46T (rs757732258), and F2015.47S (rs199609858), were identified as the most deleterious mutations that were also present in the conserved region and showed lower interactions with neighboring residues. To comprehensively understand their impact, we docked agonist dopamine and antagonist nemonapride at the binding site of the receptor, followed by 200 ns molecular dynamics simulations. We identified the V116D and I129S mutations as the most damaging, followed by F201S in the dopamine-bound states. Both the V116D and I129S variants demonstrated significantly high RMSD, Rg, and SASA, and low thermodynamic stability. The F201S-dopamine complex exhibited lower compactness and higher motions, along with a significant loss of hydrogen bonds and active site interactions. By contrast, while interacting with nemonapride, the impact of the I156T and L65P mutations was highly deleterious; both showed lower stability, higher flexibility, and higher motions. Additionally, nemonapride significantly lost interactions with the active site, notably in the I156T variant. We also found the V116D-nemonapride complex as structurally damaging; however, the interaction patterns of nemonapride were less altered in the MMPBSA analysis. Overall, this study revealed five novel deleterious variants along with a comprehensive understanding of their effect in the presence of an agonist and antagonist, which could be helpful for understanding disease susceptibility, precision medicine, and developing potential drugs.

Allostery describes the ability of biological macromolecules to transmit signals spatially through the molecule from an allosteric site – a site that is distinct from orthosteric binding sites of primary, endogenous ligands – to the functional or active site. This review starts with a historical overview and a description of the classical example of allostery – hemoglobin – and other well-known examples (aspartate transcarbamoylase, Lac repressor, kinases, G-protein-coupled receptors, adenosine triphosphate synthase, and chaperonin). We then discuss fringe examples of allostery, including intrinsically disordered proteins and inter-enzyme allostery, and the influence of dynamics, entropy, and conformational ensembles and landscapes on allosteric mechanisms, to capture the essence of the field. Thereafter, we give an overview over central methods for investigating molecular mechanisms, covering experimental techniques as well as simulations and artificial intelligence (AI)-based methods. We conclude with a review of allostery-based drug discovery, with its challenges and opportunities: with the recent advent of AI-based methods, allosteric compounds are set to revolutionize drug discovery and medical treatments.

Cyanobacteria are responsible for up to 80% of aquatic carbon dioxide fixation and have evolved a specialized carbon concentrating mechanism to increase photosynthetic yield. As such, cyanobacteria are attractive targets for synthetic biology and engineering approaches to address the demands of global energy security, food production, and climate change for an increasing world's population. The bicarbonate transporter BicA is a sodium-dependent, low-affinity, high-flux bicarbonate symporter expressed in the plasma membrane of cyanobacteria. Despite extensive biochemical characterization of BicA, including the resolution of the BicA crystal structure, the dynamic understanding of the bicarbonate transport mechanism remains elusive. To this end, we have collected over 1 ms of all-atom molecular dynamics simulation data of the BicA dimer to elucidate the structural rearrangements involved in the substrate transport process. We further characterized the energetics of the transition of BicA protomers and investigated potential mutations that are shown to decrease the free energy barrier of conformational transitions. In all, our study illuminates a detailed mechanistic understanding of the conformational dynamics of bicarbonate transporters and provides atomistic insights to engineering these transporters for enhanced photosynthetic production.

Chronic pain is a debilitating condition affecting millions worldwide, often resulting from complex interactions between the nervous and immune systems. Recent advances highlight the critical role of metabolite-sensing G protein-coupled receptors (GPCRs) in various chronic pain types. These receptors link metabolic changes with cellular responses, influencing inflammatory and degenerative processes. Receptors such as free fatty acid receptor 1 (FFAR1/GPR40), free fatty acid receptor 4 (FFAR4/GPR120), free fatty acid receptor 2 (FFAR2/GPR43), and Takeda G protein-coupled receptor 5 (TGR5/GPR131/GPBAR1) are key modulators of nociceptive signaling. GPR40, activated by long-chain fatty acids, exhibits strong anti-inflammatory effects by reducing cytokine expression. Butyrate-activated GPR43 inhibits inflammatory mediators like nitric oxide synthase-2 and cyclooxygenase-2, mitigating inflammation. TGR5, activated by bile acids, regulates inflammation and cellular senescence through pathways like NF-κB and p38. These receptors are promising therapeutic targets in chronic pain, addressing the metabolic and inflammatory factors underlying nociceptive sensitization and tissue degeneration. This review explores the molecular mechanisms of metabolite-sensing receptors in chronic pain, their therapeutic potential, and challenges in clinical application. By uncovering these mechanisms, metabolite-sensing receptors could lead to safer, more effective pain management strategies.

Somatostatin analogs, such as octreotide, lanreotide, and pasireotide, which function as somatostatin receptor ligands (SRLs), are the main drugs used for the treatment of acromegaly. These ligands are also used as important molecules for radiation therapy and imaging of neuroendocrine tumors. Somatostatin receptors (SSTRs) are canonical G protein-coupled proteins that play a role in metabolism, growth, and pathological conditions such as hormone disorders, neurological diseases, and cancers. Cryogenic electron microscopy combined with the protein structure prediction platform AlphaFold has been used to determine the 3-dimensional structures of many proteins. Recently, several groups published a series of papers illustrating the 3-dimensional structure of SSTR2, including that of the inactive/activated SSTR2-G protein complex bound to different ligands. The results revealed the residues that contribute to the ligand binding pocket and demonstrated that Trp8-Lys9 (the W-K motif) in somatostatin analogs is the key motif in stabilizing the bottom part of the binding pocket. In this review, we discuss the recent findings related to the structural analysis of SSTRs and SRLs, the relationships between the structural data and clinical findings, and the future development of novel structure-based therapies.

Activation of cannabinoid CB1 receptors (CB1R) by agonists induces analgesia but also induces cognitive impairment through the heteromer formed between CB1R and the serotonin 5HT2A receptor (5HT2AR). This side effect poses a serious drawback in the therapeutic use of cannabis for pain alleviation. Peptides designed from the transmembrane helices of CB1R, which are predicted to bind 5HT2AR and alter the stability of the CB1R-5HT2AR heteromer, have been shown to avert CB1R agonist-induced cognitive impairment while preserving analgesia. Using these peptides as templates, we have now designed nonpeptidic small molecules that prevent CB1R-5HT2AR heteromerization in bimolecular fluorescence complementation assays and the heteromerization-dependent allosteric modulations in cell signaling experiments. These results provide proof-of-principle for the design of optimized ligand-based disruptors of the CB1R-5HT2AR heteromer, opening new perspectives for in vivo studies.

Atomic-level simulations are widely used to study biomolecules and their dynamics. A common goal in such studies is to compare simulations of a molecular system under several conditions-for example, with various mutations or bound ligands-in order to identify differences between the molecular conformations adopted under these conditions. However, the large amount of data produced by simulations of ever larger and more complex systems often renders it difficult to identify the structural features that are relevant to a particular biochemical phenomenon. We present a flexible software package named Python ENSemble Analysis (PENSA) that enables a comprehensive and thorough investigation into biomolecular conformational ensembles. It provides featurization and feature transformations that allow for a complete representation of biomolecules such as proteins and nucleic acids, including water and ion binding sites, thus avoiding the bias that would come with manual feature selection. PENSA implements methods to systematically compare the distributions of molecular features across ensembles to find the significant differences between them and identify regions of interest. It also includes a novel approach to quantify the state-specific information between two regions of a biomolecule, which allows, for example, tracing information flow to identify allosteric pathways. PENSA also comes with convenient tools for loading data and visualizing results, making them quick to process and easy to interpret. PENSA is an open-source Python library maintained at https://github.com/drorlab/pensa along with an example workflow and a tutorial. We demonstrate its usefulness in real-world examples by showing how it helps us determine molecular mechanisms efficiently.

Opioid receptors, a subfamily of G protein-coupled receptors (GPCRs), are key therapeutic targets. In the canonical GPCR activation model, agonist binding is required for receptor-G protein complex formation, while antagonists prevent G protein coupling. However, many GPCRs exhibit basal activity, allowing G protein association without an agonist. The pharmacological impact of agonist-free receptor-G protein complexes is poorly understood. Here we present biochemical evidence that certain κ-opioid receptor (KOR) inverse agonists can act via KOR-Gi protein complexes. To investigate this phenomenon, we determined cryo-EM structures of KOR-Gi protein complexes with three inverse agonists: JDTic, norBNI and GB18, corresponding to structures of inverse agonist-bound GPCR-G protein complexes. Remarkably, the orthosteric binding pocket resembles the G protein-free 'inactive' receptor conformation, while the receptor remains coupled to the G protein. In summary, our work challenges the canonical model of receptor antagonism and offers crucial insights into GPCR pharmacology.

Negative allosteric modulation of G-protein coupled receptors (GPCRs) by Na+ ions was first described in the 1970s for opioid receptors (ORs) and has subsequently been detected for most class A GPCRs. In high-resolution structures of inactive-state class A GPCRs, a Na+ ion binds to a conserved pocket near residue D2.50, whereas active-state structures of GPCRs are incompatible with Na+ binding. Correspondingly, Na+ diminishes agonist affinity, stabilizes the receptors in the inactive state, and reduces basal signaling. We applied a mutual-information based analysis to μs-timescale biomolecular simulations of the μ-opioid receptor (μ-OR). Our results reveal that Na+ binding is coupled to a water wire linking the Na+ binding site with the agonist binding pocket and to rearrangements in polar networks propagating conformational changes to the agonist and G-protein binding sites. These findings provide a new mechanistic link between the presence of the ion, altered agonist affinity, receptor deactivation, and lowered basal signaling levels.