What are G protein-coupled receptors?
G protein-coupled receptors, or GPCRs, are a critical class of membrane protein responsible for transporting chemical signals from the extracellular matrix into the cell. There are hundreds of distinct GPCRs encoded by the human genome, specific to a variety of signaling molecules from ions to small proteins. Due to their ubiquitous nature, GPCRs have been associated with a wide variety of diseases and are actively being investigated as drug targets by the pharmaceutical industry.
Despite the promise of GPCRs, we only know a small fraction of their structures and most remain undruggable. This is due to the inherent difficulty of crystallizing and analyzing membrane proteins, which often necessitate complex sample preparation in order to produce crystals of appropriate size for crystallography.
Cryo-EM of GPCR structures
Cryo-electron microscopy (cryo-EM) is an increasingly popular life science technique due to its ability to generate high-resolution 3D structures for complex biological samples without the need for crystallization. This makes cryo-EM ideally suited for the analysis of membrane proteins, such as GPCRs, and there is a rapidly increasing number of cryo-EM membrane protein structures in the protein databank. Not only are these structures high-resolution, but they are often corroborated by multiple research groups, pointing to the reliability and utility of cryo-EM for this kind of analysis.
Benefits of cryo-EM analysis of GPCRs
Beyond enabling structure-based drug design for a wider range of GPCRs, cryo-EM has additional advantages that make it a promising component of the drug discovery process.
Most critically, cryo-EM provides active state structures for GPCRs, including low-affinity states. Even at low resolutions this can provide valuable insight into binding sites and can assist in modeling efforts. Research groups have successfully used cryo-EM to observe conformational rearrangements, to pinpoint drug-receptor interactions, and to investigate the specificity of Gs- and Gi-class G-proteins.
It is worth noting that all these observations were made early in the drug discovery process, enabling pharmaceutical companies to use structural insights to guide drug design.
Cryo-electron microscopes for GPCR structure analysis
Cryo-EM instrumentation has become significantly more approachable in recent years. What was once a highly specialized instrument requiring a dedicated expert operator can now be used by any researcher thanks to a bevy of ease-of-use and automation improvements.
The Thermo Scientific Glacios Cryo-TEM has reliably produced ~3.2 Å structures, whereas the higher-resolution Thermo Scientific Krios Cryo-TEM regularly generates ~2 Å resolution data. On top of that, automation improvements have reduced throughput time so that entirely novel structures can be obtained in as little as one week. This makes Thermo Scientific cryo-EM instrumentation ideally suited for the fast pace of pharmaceutical research, providing high-quality analysis for structure-based drug design.
Case study: GLP1R
Glucagon-like peptide-1 receptor (GLP1R) is a critical pharmacological target in the treatment of metabolic disorders. In fact, peptides that mimic the function of GLP-1 (e.g. semaglutide or liraglutide) are already being prescribed for the treatment of type-2 diabetes and for chronic weight management. These peptides are currently administered via injection; structural characterization of their binding sites would allow for the development of small molecule alternatives that could be administered orally.
Highlighting the accessibility of cryo-EM, multiple research groups have already used this technique to determine the structure of GLP1R at sufficient resolution to visualize not just the binding pocket but even hydrogen bonds and water molecules. These structural insights are accelerating lead optimization by showing how the molecular machinery of the peptide is impacting downstream signaling.
Single Particle Analysis
Single particle analysis (SPA) is a cryo-electron microscopy technique that enables structural characterization at near-atomic resolutions, unraveling dynamic biological processes and the structure of biomolecular complexes/assemblies.
MicroED
MicroED is an exciting new technique with applications in the structural determination of small molecules and protein. With this method, atomic details can be extracted from individual nanocrystals (<200 nm in size), even in a heterogeneous mixture.
Single Particle Analysis
Single particle analysis (SPA) is a cryo-electron microscopy technique that enables structural characterization at near-atomic resolutions, unraveling dynamic biological processes and the structure of biomolecular complexes/assemblies.
MicroED
MicroED is an exciting new technique with applications in the structural determination of small molecules and protein. With this method, atomic details can be extracted from individual nanocrystals (<200 nm in size), even in a heterogeneous mixture.
Proteins Analysis
Cryo-electron microscopy provides near-atomic resolution 3D protein structure. It can determine structural information for complexes and crystallization-resistant samples, as well as vital cellular context.
Biopharmaceutical Research
Structural drug discovery is enabled by cryo-electron microscopy, as the method provides near-atomic-resolution detail for small molecules and protein biologics in their fully hydrated state.
Virus Analysis
Cryo-EM enables the 3D structural visualization of virus particles, and the antigen-antibody interface, at near-atomic resolutions. A virus’s inherent structural symmetry makes it the ideal target for cryo-EM analysis.
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