ABSTRACT
Class B G protein-coupled receptors (GPCRs) are important therapeutic targets for major diseases. Here, we present structures of peptide and Gs-bound pituitary adenylate cyclase-activating peptide, PAC1 receptor, and corticotropin-releasing factor (CRF), (CRF1) receptor. Together with recently solved structures, these provide coverage of the major class B GPCR subfamilies. Diverse orientations of the extracellular domain to the receptor core in different receptors are at least partially dependent on evolutionary conservation in the structure and nature of peptide interactions. Differences in peptide interactions to the receptor core also influence the interlinked TM2-TM1-TM6/ECL3/TM7 domain, and this is likely important in their diverse signaling. However, common conformational reorganization of ECL2, linked to reorganization of ICL2, modulates G protein contacts. Comparison between receptors reveals ICL2 as a key domain forming dynamic G protein interactions in a receptor- and ligand-specific manner. This work advances our understanding of class B GPCR activation and Gs coupling.
Subject(s)
Receptors, Corticotropin-Releasing Hormone/ultrastructure , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide/ultrastructure , Amino Acid Sequence , Cryoelectron Microscopy/methods , Enkephalins , Humans , Ligands , Models, Molecular , Peptides , Protein Precursors , Receptors, Corticotropin-Releasing Hormone/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/ultrastructure , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide/metabolism , Signal TransductionABSTRACT
Peptide drugs targeting class B1 G-protein-coupled receptors (GPCRs) can treat multiple diseases; however, there remains substantial interest in the development of orally delivered non-peptide drugs. Here, we reveal unexpected overlap between signaling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist PF 06882961 and GLP-1 that was not observed for another compound, CHU-128. Compounds from these patent series, including PF 06882961, are currently in clinical trials for treatment of type 2 diabetes. High-resolution cryoelectron microscopy (cryo-EM) structures reveal that the binding sites for PF 06882961 and GLP-1 substantially overlap, whereas CHU-128 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not CHU-128, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of non-peptide agonists targeting class B GPCRs.
Subject(s)
Glucagon-Like Peptide-1 Receptor/agonists , Glucagon-Like Peptide-1 Receptor/metabolism , Animals , Binding Sites/physiology , Cryoelectron Microscopy/methods , Glucagon-Like Peptide 1/chemistry , Glucagon-Like Peptide 1/metabolism , Glucagon-Like Peptide-1 Receptor/chemistry , Humans , Peptides/chemistry , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Structure-Activity RelationshipABSTRACT
In situ cryo-electron tomography enables investigation of macromolecules in their native cellular environment. Samples have become more readily available owing to recent software and hardware advancements. Data collection, however, still requires an experienced operator and appreciable microscope time to carefully select targets for high-throughput tilt series acquisition. Here, we developed smart parallel automated cryo-electron tomography (SPACEtomo), a workflow using machine learning approaches to fully automate the entire cryo-electron tomography process, including lamella detection, biological feature segmentation, target selection and parallel tilt series acquisition, all without the need for human intervention. This degree of automation will be essential for obtaining statistically relevant datasets and high-resolution structures of macromolecules in their native context.
ABSTRACT
The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.
Subject(s)
Analgesia , Receptor, Adenosine A1/metabolism , Adenosine/chemistry , Adenosine/metabolism , Allosteric Regulation/drug effects , Analgesia/methods , Animals , Binding Sites , Disease Models, Animal , Female , GTP-Binding Protein alpha Subunit, Gi2/chemistry , GTP-Binding Protein alpha Subunit, Gi2/metabolism , Hyperalgesia/drug therapy , Lipids , Male , Neuralgia/drug therapy , Neuralgia/metabolism , Protein Stability/drug effects , Rats , Rats, Sprague-Dawley , Receptor, Adenosine A1/chemistry , Signal Transduction/drug effectsABSTRACT
In situ cryo electron tomography of cryo focused ion beam milled samples has emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. However, the possibilities for recording tomographic tilt series in a high-throughput manner are limited, in part by the lamella-shaped samples. Here we utilize a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining access to sample areas conventionally used for tracking specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows for the collection of sample areas that were previously unreachable, thus maximizing the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high throughput and quality of PACE-tomo.
Subject(s)
Electron Microscope Tomography , Ribosomes , Electron Microscope Tomography/methods , Cryoelectron Microscopy/methods , Macromolecular Substances/chemistryABSTRACT
Amylin receptors (AMYRs), heterodimers of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins, are promising obesity targets. A hallmark of AMYR activation by Amy is the formation of a 'bypass' secondary structural motif (residues S19-P25). This study explored potential tuning of peptide selectivity through modification to residues 19-22, resulting in a selective AMYR agonist, San385, as well as nonselective dual amylin and calcitonin receptor agonists (DACRAs), with San45 being an exemplar. We determined the structure and dynamics of San385-bound AMY3R, and San45 bound to AMY3R or CTR. San45, via its conjugated lipid at position 21, was anchored at the edge of the receptor bundle, enabling a stable, alternative binding mode when bound to the CTR, in addition to the bypass mode of binding to AMY3R. Targeted lipid modification may provide a single intervention strategy for design of long-acting, nonselective, Amy-based DACRAs with potential anti-obesity effects.
Subject(s)
Islet Amyloid Polypeptide , Receptors, Calcitonin , Humans , Receptors, Calcitonin/agonists , Receptors, Calcitonin/metabolism , Islet Amyloid Polypeptide/metabolism , Obesity , LipidsABSTRACT
Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2-6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.
Subject(s)
Glucagon-Like Peptide-1 Receptor/agonists , Isoquinolines/pharmacology , Phenylalanine/analogs & derivatives , Pyridines/pharmacology , Animals , CHO Cells , Cricetinae , Cricetulus , Glucagon-Like Peptide-1 Receptor/chemistry , Glucagon-Like Peptide-1 Receptor/metabolism , Humans , Isoquinolines/chemistry , Kinetics , Models, Molecular , Phenylalanine/chemistry , Phenylalanine/pharmacology , Protein Structure, Quaternary , Protein Structure, Tertiary , Pyridines/chemistry , Structural Homology, ProteinABSTRACT
Inhibition of calcitonin gene-related peptide (CGRP) or its cognate CGRP receptor (CGRPR) has arisen as a major breakthrough in the treatment of migraine. However, a second CGRP-responsive receptor exists, the amylin (Amy) 1 receptor (AMY1R), yet its involvement in the pathology of migraine is poorly understood. AMY1R and CGRPR are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CLR), respectively. Here, we present the structure of AMY1R in complex with CGRP and Gs protein and compare it with the reported structures of the AMY1R complex with rat amylin (rAmy) and the CGRPR in complex with CGRP. Despite similar protein backbones observed within the receptors and the N- and C-termini of the two peptides bound to the AMY1R complexes, they have distinct organization in the peptide midregions (the bypass motif) that is correlated with differences in the dynamics of the respective receptor extracellular domains. Moreover, divergent conformations of extracellular loop (ECL) 3, intracellular loop (ICL) 2, and ICL3 within the CTR and CLR protomers are evident when comparing the CGRP bound to the CGRPR and AMY1R, which influences the binding mode of CGRP. However, the conserved interactions made by the C-terminus of CGRP to the CGRPR and AMY1R are likely to account for cross-reactivity of nonpeptide CGRPR antagonists observed at AMY1R, which also extends to other clinically used CGRPR blockers, including antibodies.
Subject(s)
Calcitonin Gene-Related Peptide , Cryoelectron Microscopy , Receptor Activity-Modifying Protein 1 , Humans , Calcitonin Gene-Related Peptide/metabolism , Calcitonin Gene-Related Peptide/chemistry , Receptor Activity-Modifying Protein 1/metabolism , Receptor Activity-Modifying Protein 1/chemistry , Receptors, Islet Amyloid Polypeptide/metabolism , Receptors, Islet Amyloid Polypeptide/chemistry , Animals , Rats , Models, Molecular , Receptors, Calcitonin Gene-Related Peptide/metabolism , Receptors, Calcitonin Gene-Related Peptide/chemistry , Protein ConformationABSTRACT
Recent advances in G-protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multidimensional signal propagation mediated by these receptors depends, in part, on their conformational mobility; however, the relationship between receptor function and static structures is inherently uncertain. Here, we examine the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide 1 receptor (GLP-1R), an important clinical target. We use variants of the peptides GLP-1 and exendin-4 (Ex4) to explore the interplay between helical propensity near the agonist N terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an Ex4 analog, the GLP-1R and Gs heterotrimer revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data, along with molecular dynamics (MD) simulations, suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.
Subject(s)
Glucagon-Like Peptide 1 , Glucagon-Like Peptide-1 Receptor , Exenatide , Glucagon-Like Peptide 1/metabolism , Glucagon-Like Peptide-1 Receptor/chemistry , Peptides/chemistry , Protein DomainsABSTRACT
G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from "unwinding" of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs.
Subject(s)
GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , GTP-Binding Protein alpha Subunits, Gs/metabolism , Receptors, Cholecystokinin/chemistry , Receptors, Cholecystokinin/metabolism , Cholecystokinin/metabolism , Cholesterol/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/chemistry , GTP-Binding Protein alpha Subunits, Gq-G11/ultrastructure , GTP-Binding Protein alpha Subunits, Gs/chemistry , GTP-Binding Protein alpha Subunits, Gs/ultrastructure , HEK293 Cells , Humans , Models, Molecular , Protein Binding , Receptors, Cholecystokinin/ultrastructure , Signal TransductionABSTRACT
The class A adenosine A1 receptor (A1R) is a G-protein-coupled receptor that preferentially couples to inhibitory Gi/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A1R in complex with adenosine and heterotrimeric Gi2 protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive A1R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A1R primarily via amino acids in the C terminus of the Gαi α5-helix, concomitant with a 10.5 Å outward movement of the A1R transmembrane domain 6. Comparison with the agonist-bound ß2 adrenergic receptor-Gs-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A1R structure provides molecular insights into receptor and G-protein selectivity.
Subject(s)
Adenosine/chemistry , Adenosine/metabolism , Cryoelectron Microscopy , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/ultrastructure , Receptor, Adenosine A1/chemistry , Receptor, Adenosine A1/ultrastructure , Binding Sites , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gs/chemistry , GTP-Binding Protein alpha Subunits, Gs/metabolism , Humans , Models, Molecular , Receptor, Adenosine A1/metabolism , Rotation , Substrate SpecificityABSTRACT
The class B glucagon-like peptide-1 (GLP-1) G protein-coupled receptor is a major target for the treatment of type 2 diabetes and obesity. Endogenous and mimetic GLP-1 peptides exhibit biased agonism-a difference in functional selectivity-that may provide improved therapeutic outcomes. Here we describe the structure of the human GLP-1 receptor in complex with the G protein-biased peptide exendin-P5 and a Gαs heterotrimer, determined at a global resolution of 3.3 Å. At the extracellular surface, the organization of extracellular loop 3 and proximal transmembrane segments differs between our exendin-P5-bound structure and previous GLP-1-bound GLP-1 receptor structure. At the intracellular face, there was a six-degree difference in the angle of the Gαs-α5 helix engagement between structures, which was propagated across the G protein heterotrimer. In addition, the structures differed in the rate and extent of conformational reorganization of the Gαs protein. Our structure provides insights into the molecular basis of biased agonism.
Subject(s)
Cryoelectron Microscopy , GTP-Binding Protein alpha Subunits, Gs/chemistry , GTP-Binding Protein alpha Subunits, Gs/ultrastructure , Glucagon-Like Peptide 1/chemistry , Glucagon-Like Peptide 1/pharmacology , Glucagon-Like Peptide-1 Receptor/agonists , Glucagon-Like Peptide-1 Receptor/ultrastructure , Binding Sites , Glucagon-Like Peptide 1/metabolism , Glucagon-Like Peptide-1 Receptor/chemistry , Humans , Models, Molecular , Protein ConformationABSTRACT
Cryo-electron microscopy (cryo-EM) has emerged as a powerful structure determination technique. Its most prolific branch is single particle analysis (SPA), a method being used in a growing number of laboratories worldwide to determine high-resolution protein structures. Cryo-electron tomography (cryo-ET) is another powerful approach that enables visualization of protein complexes in their native cellular environment. Despite the wide-ranging success of cryo-EM, there are many methodological aspects that could be improved. Those include sample preparation, sample screening, data acquisition, image processing, and structure validation. Future developments will increase the reliability and throughput of the technique and reduce the cost and skill level barrier for its adoption.
Subject(s)
Cryoelectron Microscopy , Proteins/chemistry , Protein Conformation , Proteins/metabolismABSTRACT
Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gαsßγ protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the Gß subunit. This structure provides a new framework for understanding G-protein-coupled receptor function.
Subject(s)
Cryoelectron Microscopy , Heterotrimeric GTP-Binding Proteins/metabolism , Heterotrimeric GTP-Binding Proteins/ultrastructure , Receptors, Calcitonin/classification , Receptors, Calcitonin/ultrastructure , Binding Sites , Cell Membrane/metabolism , Conserved Sequence , Heterotrimeric GTP-Binding Proteins/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Models, Molecular , Protein Conformation , Receptors, Calcitonin/agonists , Receptors, Calcitonin/metabolismABSTRACT
The histone H3 variant CENP-A marks centromeres epigenetically and is essential for mitotic fidelity. Previous crystallographic studies of the CENP-A nucleosome core particle (NCP) reconstituted with a human α-satellite DNA derivative revealed both DNA ends to be highly flexible, a feature important for CENP-A mitotic functions. However, recent cryo-EM studies of CENP-A NCP complexes comprising primarily Widom 601 DNA reported well-ordered DNA ends. Here, we report the cryo-EM structure of the CENP-A 601 NCP determined by Volta phase-plate imaging. The data reveal that one ('left') 601 DNA end is well ordered whereas the other ('right') end is flexible and partly detached from the histone core, suggesting sequence-dependent dynamics of the DNA termini. Indeed, a molecular dynamics simulation of the CENP-A 601 NCP confirmed the distinct dynamics of the two DNA extremities. Reprocessing the image data using two-fold symmetry yielded a cryo-EM map in which both DNA ends appeared well ordered, indicating that such an artefact may inadvertently arise if NCP asymmetry is lost during image processing. These findings enhance our understanding of the dynamic features that discriminate CENP-A from H3 nucleosomes by revealing that DNA end flexibility can be fine-tuned in a sequence-dependent manner.
Subject(s)
Centromere Protein A/chemistry , DNA/chemistry , Nucleosomes/chemistry , Cryoelectron Microscopy , Humans , Molecular Dynamics Simulation , Nucleosomes/ultrastructureABSTRACT
Proteasomes occur in all three domains of life, and are the principal molecular machines for the regulated degradation of intracellular proteins. They play key roles in the maintenance of protein homeostasis, and control vital cellular processes. While the eukaryotic 26S proteasome is extensively characterized, its putative evolutionary precursor, the archaeal proteasome, remains poorly understood. The primordial archaeal proteasome consists of a 20S proteolytic core particle (CP), and an AAA-ATPase module. This minimal complex degrades protein unassisted by non-ATPase subunits that are present in a 26S proteasome regulatory particle (RP). Using cryo-EM single-particle analysis, we determined structures of the archaeal CP in complex with the AAA-ATPase PAN (proteasome-activating nucleotidase). Five conformational states were identified, elucidating the functional cycle of PAN, and its interaction with the CP. Coexisting nucleotide states, and correlated intersubunit signaling features, coordinate rotation of the PAN-ATPase staircase, and allosterically regulate N-domain motions and CP gate opening. These findings reveal the structural basis for a sequential around-the-ring ATPase cycle, which is likely conserved in AAA-ATPases.
Subject(s)
Adenosine Triphosphatases/ultrastructure , Archaeal Proteins/ultrastructure , Archaeoglobus fulgidus/enzymology , Cryoelectron Microscopy , Proteasome Endopeptidase Complex/ultrastructureABSTRACT
Unimolecular dual agonists of the glucagon (GCG) receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) are a new class of drugs that are potentially superior to GLP-1R-specific agonists for the management of metabolic disease. The dual-agonist, peptide 15 (P15), is a glutamic acid 16 analog of GCG with GLP-1 peptide substitutions between amino acids 17 and 24 that has potency equivalent to those of the cognate peptide agonists at the GCGR and GLP-1R. Here, we have used cryo-EM to solve the structure of an active P15-GCGR-Gs complex and compared this structure to our recently published structure of the GCGR-Gs complex bound to GCG. This comparison revealed that P15 has a reduced interaction with the first extracellular loop (ECL1) and the top of transmembrane segment 1 (TM1) such that there is increased mobility of the GCGR extracellular domain and at the C terminus of the peptide compared with the GCG-bound receptor. We also observed a distinct conformation of ECL3 and could infer increased mobility of the far N-terminal His-1 residue in the P15-bound structure. These regions of conformational variance in the two peptide-bound GCGR structures were also regions that were distinct between GCGR structures and previously published peptide-bound structures of the GLP-1R, suggesting that greater conformational dynamics may contribute to the increased efficacy of P15 in activation of the GLP-1R compared with GCG. The variable domains in this receptor have previously been implicated in biased agonism at the GLP-1R and could result in altered signaling of P15 at the GCGR compared with GCG.
Subject(s)
Cryoelectron Microscopy , Peptides/chemistry , Receptors, Glucagon , Animals , Glucagon-Like Peptide-1 Receptor/agonists , Glucagon-Like Peptide-1 Receptor/chemistry , Glucagon-Like Peptide-1 Receptor/ultrastructure , Humans , Protein Domains , Protein Structure, Quaternary , Receptors, Glucagon/agonists , Receptors, Glucagon/chemistry , Receptors, Glucagon/ultrastructureABSTRACT
Dual agonists that can activate both the glucagon-like peptide-1 receptor (GLP-1R) and the gastric inhibitory polypeptide receptor (GIPR) have demonstrated high efficacy for the treatment of metabolic disease. Peptide-19 is a prototypical dual agonist that has high potency at both GLP-1R and GIPR but has a distinct signalling profile relative to the native peptides at the cognate receptors. In this study, we solved the structure of peptide-19 bound to the GLP-1R in complex with Gs protein, and compared the structure and dynamics of this complex to that of published structures of GLP-1R:Gs in complex with other receptor agonists. Unlike other peptide-bound receptor complexes, peptide-19:GLP-1R:Gs demonstrated a more open binding pocket where transmembrane domain (TM) 6, TM7 and the interconnecting extracellular loop 3 (ECL3) were located away from the peptide, with no interactions between peptide-19 and TM6/ECL3. Analysis of conformational variance of the complex revealed that peptide-19 was highly dynamic and underwent binding and unbinding motions facilitated by the more open TM binding pocket. Both the consensus structure of the GLP-1R complex with peptide-19 and the dynamics of this complex were distinct from previously described GLP-1R structures providing unique insights into the mode of GLP-1R activation by this dual agonist.
Subject(s)
Glucagon-Like Peptide-1 Receptor/chemistry , Glucagon-Like Peptide-1 Receptor/metabolism , Peptides/chemistry , Peptides/metabolism , Cryoelectron Microscopy/methods , Glucagon-Like Peptide-1 Receptor/agonists , Protein Domains , Protein Structural ElementsABSTRACT
Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca2+-triggered release.
Subject(s)
Membrane Fusion Proteins/metabolism , Membrane Fusion , Neurons/metabolism , Synaptic Membranes/metabolism , Animals , Calcium/metabolism , Cryoelectron Microscopy/methods , Membrane Fusion Proteins/chemistry , Models, Molecular , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Protein Binding , Protein Domains , Proteolipids/metabolism , Proteolipids/ultrastructure , SNARE Proteins/chemistry , SNARE Proteins/metabolism , Synaptic Membranes/ultrastructure , Synaptic Vesicles/metabolism , Synaptic Vesicles/ultrastructure , Synaptotagmin I/chemistry , Synaptotagmin I/metabolismABSTRACT
The power of cryo-electron tomography (cryoET) lies in its capability to characterize macromolecules in their cellular context. Structure determination by cryoET, however, is time-consuming compared to single particle approaches. A recent study reported significant acceleration of data acquisition by a fast-incremental single-exposure (FISE) tilt series scheme. Here we improved the method and evaluated its efficiency and performance. We show that (1) FISE combined with the latest generation of direct electron detectors speeds up collection considerably, (2) previous generation (pre-2017) double-tilt axis Titan Krios holders are also suitable for FISE data acquisition, (3) x, y and z-specimen shifts can be compensated for, and (4) FISE tilt series data can generate averages of sub-nanometer resolution. These advances will allow for a widespread adoption of cryoET for high-throughput in situ studies and high-resolution structure determination across different biological research disciplines.