Project description:The ?1 receptor is a poorly understood membrane protein expressed throughout the human body. Ligands targeting the ?1 receptor are in clinical trials for treatment of Alzheimer's disease, ischemic stroke, and neuropathic pain. However, relatively little is known regarding the ?1 receptor's molecular function. Here, we present crystal structures of human ?1 receptor bound to the antagonists haloperidol and NE-100, and the agonist (+)-pentazocine, at crystallographic resolutions of 3.1?Å, 2.9?Å, and 3.1?Å, respectively. These structures reveal a unique binding pose for the agonist. The structures and accompanying molecular dynamics (MD) simulations identify agonist-induced structural rearrangements in the receptor. Additionally, we show that ligand binding to ?1 is a multistep process that is rate limited by receptor conformational change. We used MD simulations to reconstruct a ligand binding pathway involving two major conformational changes. These data provide a framework for understanding the molecular basis for ?1 agonism.
Project description:The leukotriene B4 receptor 1 (BLT1) regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious, allergic, metabolic, and tumorigenic human diseases. Here we present a crystal structure of human BLT1 (hBLT1) in complex with a selective antagonist MK-D-046, developed for the treatment of type 2 diabetes and other inflammatory conditions. Comprehensive analysis of the structure and structure-activity relationship data, reinforced by site-directed mutagenesis and docking studies, reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species. The structure helps to identify a putative membrane-buried ligand access channel as well as potential receptor binding modes of endogenous agonists. These structural insights of hBLT1 enrich our understanding of its ligand recognition and open up future avenues in structure-based drug design.
Project description:The small cyclic neuropeptide hormone oxytocin (OT) and its cognate receptor play a central role in the regulation of social behaviour and sexual reproduction. Here we report the single-particle cryo-electron microscopy structure of the active oxytocin receptor (OTR) in complex with its cognate ligand oxytocin. Our structure provides high-resolution insights into the OT binding mode, the OTR activation mechanism as well as the subtype specificity within the oxytocin/vasopressin receptor family.
Project description:The CC chemokine receptor 7 (CCR7) balances immunity and tolerance by homeostatic trafficking of immune cells. In cancer, CCR7-mediated trafficking leads to lymph node metastasis, suggesting the receptor as a promising therapeutic target. Here, we present the crystal structure of human CCR7 fused to the protein Sialidase NanA by using data up to 2.1 Å resolution. The structure shows the ligand Cmp2105 bound to an intracellular allosteric binding pocket. A sulfonamide group, characteristic for various chemokine receptor ligands, binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix 7 and helix 8. We demonstrate how structural data can be used in combination with a compound repository and automated thermal stability screening to identify and modulate allosteric chemokine receptor antagonists. We detect both novel (CS-1 and CS-2) and clinically relevant (CXCR1-CXCR2 phase-II antagonist Navarixin) CCR7 modulators with implications for multi-target strategies against cancer.
Project description:Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.
Project description:Hydroxycarboxylic acid receptor 2 (HCAR2) belongs to the family of class A G protein-coupled receptors with key roles in regulating lipolysis and free fatty acid formation in humans. It is deeply involved in many pathophysiological processes and serves as an attractive target for the treatment of cardiovascular, neoplastic, autoimmune, neurodegenerative, inflammatory, and metabolic diseases. Here, we report four cryo-EM structures of human HCAR2-Gi1 complexes with or without agonists, including the drugs niacin (2.69 Å) and acipimox (3.23 Å), the highly subtype-specific agonist MK-6892 (3.25 Å), and apo form (3.28 Å). Combined with molecular dynamics simulation and functional analysis, we have revealed the recognition mechanism of HCAR2 for different agonists and summarized the general pharmacophore features of HCAR2 agonists, which are based on three key residues R1113.36, S17945.52, and Y2847.43. Notably, the MK-6892-HCAR2 structure shows an extended binding pocket relative to other agonist-bound HCAR2 complexes. In addition, the key residues that determine the ligand selectivity between the HCAR2 and HCAR3 are also illuminated. Our findings provide structural insights into the ligand recognition, selectivity, activation, and G protein coupling mechanism of HCAR2, which shed light on the design of new HCAR2-targeting drugs for greater efficacy, higher selectivity, and fewer or no side effects.
Project description:Atherosclerosis is a chronic inflammatory disorder of cholesterol deposition in monocyte-derived macrophages (MDM) within the arterial wall leading to impingement on the lumen of the vessel. In atherosclerotic lesions, MDM are the primary source of NADPH oxidase-derived superoxide anion (O??) inducing low-density lipoprotein (LDL) oxidation leading to their unregulated uptake of oxidized LDL and foam cell formation. We recently discovered that zymosan potently activates monocyte NADPH oxidase via the non-toll pattern recognition receptor (PRR), Dectin-1. Other PRRs bind endogenous human ligands, yet no such ligands have been identi?ed for Dectin-1. Our hypothesis was that in?ammation generates endogenous ligands for Dectin-1 that activate O?? production and thereby contributes to atherogenesis.Human: anti-zymosan antibodies were used to identify similar, cross-reactive epitopes in human atherosclerotic tissue extracts. Immunoblot analysis revealed consistent antibody reactive protein bands on one- and two-dimensional gel electrophoreses. Vimentin was identified by mass spectrometry in the immunoreactive bands across different tissue samples. Direct binding of vimentin to Dectin-1 was observed using BIACORE. Further data revealed that vimentin induces O?? production by human monocytes. Analysis of human atherosclerotic lesions revealed that vimentin was detected extracellularly in the necrotic core and in areas of active inflammation. Vimentin also co-localized with Dectin-1 in macrophage-rich regions where O?? is produced.We conclude that vimentin is an endogenous, activating ligand for Dectin-1. Its presence in areas of artery wall inflammation and O?? production suggests that vimentin activates Dectin-1 and contributes to the oxidation of lipids and cholesterol accumulation in atherosclerosis.
Project description:Lysophosphatidylserine (LysoPS) is a lipid mediator that induces multiple cellular responses through binding to GPR174. Here, we present the cryo-electron microscopy (cryo-EM) structure of LysoPS-bound human GPR174 in complex with Gs protein. The structure reveals a ligand recognition mode, including the negatively charged head group of LysoPS forms extensive polar interactions with surrounding key residues of the ligand binding pocket, and the L-serine moiety buries deeply into a positive charged cavity in the pocket. In addition, the structure unveils a partially open pocket on transmembrane domain helix (TM) 4 and 5 for a lateral entry of ligand. Finally, the structure reveals a Gs engaging mode featured by a deep insertion of a helix 5 (αH5) and extensive polar interactions between receptor and αH5. Taken together, the information revealed by our structural study provides a framework for understanding LysoPS signaling and a rational basis for designing LysoPS receptor-targeting drugs.
Project description:Angiotensin converting enzyme 2 (ACE2) plays a key role in renin-angiotensin system regulation and amino acid homeostasis. Human ACE2 acts as the receptor for severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2. ACE2 is also widely expressed in epithelial cells of lungs, heart, kidney and pancreas. It is considered an important drug target for treating SARS-CoV-2, as well as pulmonary diseases, heart failure, hypertension, renal diseases and diabetes. Despite the critical importance, the mechanism of ligand binding to the human ACE2 receptor remains unknown. Here, we address this challenge through all-atom simulations using a novel ligand Gaussian accelerated molecular dynamics (LiGaMD) method. Microsecond LiGaMD simulations have successfully captured both binding and unbinding of the MLN-4760 inhibitor in the ACE2 receptor. In the ligand unbound state, the ACE2 receptor samples distinct Open, Partially Open and Closed conformations. Ligand binding biases the receptor conformational ensemble towards the Closed state. The LiGaMD simulations thus suggest a conformational selection mechanism for ligand recognition by the ACE2 receptor. Our simulation findings are expected to facilitate rational drug design of ACE2 against coronaviruses and other related human diseases.
Project description:Angiotensin converting enzyme 2 (ACE2) plays a key role in renin-angiotensin system regulation and amino acid homeostasis. Human ACE2 acts as the receptor for severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2. ACE2 is also widely expressed in epithelial cells of the lungs, heart, kidney, and pancreas. It is considered an important drug target for treating SARS-CoV-2 as well as pulmonary diseases, heart failure, hypertension, renal diseases, and diabetes. Despite the critical importance, the mechanism of ligand binding to the human ACE2 receptor remains unknown. Here, we have addressed this challenge through all-atom simulations using a novel ligand Gaussian accelerated molecular dynamics (LiGaMD) method. Microsecond time scale LiGaMD simulations have unprecedentedly captured multiple times of spontaneous binding and unbinding of a potent inhibitor MLN-4760 in the ACE2 receptor. With ligand far away in the unbound state, the ACE2 receptor samples distinct Open, Partially Open, Closed, and Fully Closed conformations. Upon ligand binding to the active site, conformational ensemble of the ACE2 receptor is biased toward the Closed state as observed in the X-ray experimental structure. The LiGaMD simulations thus suggest a conformational selection mechanism for ligand recognition by the highly flexible ACE2 receptor, which is expected to facilitate rational drug design targeting human ACE2 against coronaviruses and other related human diseases.