Human cells express over 800 G-protein-coupled receptors (GPCRs) to facilitate communication with the external environment. These receptors respond to a variety of signals by undergoing structural changes that activate internal G proteins, β-arrestins, and other transducers1. Capturing the dynamics of GPCR activation has always been a challenge because G protein activation in cells occurs in less than a second, reflecting the transient nature of these active states. A recent breakthrough study published in Nature by Makaía M. Papasergi-Scott and colleagues has made significant progress in this area 2. Using time-resolved cryo-electron microscopy (cryo-EM) and variability analysis to monitor the transitions of the Gs protein in complex with the β2-adrenergic receptor (β2AR) at brief sequential intervals following GTP addition, the research team identified the conformational changes that underlie G protein activation and its separation from the receptor. Twenty transition structures generated from overlapping particle subsets along this pathway provide a high-resolution description of the events driving G protein activation upon GTP binding. Unlike previous studies that provided only static snapshots, this approach captures the entire activation sequence in vivid detail.
The captured structures reveal a dynamic of conformational changes initiated by the binding of an agonist isoproterenol to β2AR. This interaction significantly increases the receptor’s affinity for GTP, allowing a detailed observation of its interaction with the Gs protein in its activated state. From the initial GTP binding, the structures highlight critical shifts in the α5 helix and the α-helical domain (AHD) of the G protein. These alterations are essential for the complex mechanism of G protein activation, following GTP association with the Gα NTP binding pocket.
The activation process begins with the α-helical domain (AHD) of the G protein in an open state, which is crucial for the initial binding of GTP. This early interaction sets the stage for a cascade of significant conformational changes. As GTP stabilizes and migrates towards the P loop, it causes the TCAT motif to shift, a key movement for the subsequent closure of the AHD. Concurrently, the α1 helix extends, propagating structural changes throughout the G protein. Further changes include the movement of Switch II towards the nucleotide-binding pocket and the stabilization of Switch III, both integral to the activation of the G protein. The AHD transitioning to a closed conformation prompts the β2–β3 loop to move away from the α5 helix, disrupting an ionic lock and allowing the α5 helix to start transitioning. This helix undergoes significant restructuring—it breaks and reforms closer to the TCAT motif, which is crucial for the final steps of G protein activation.
This detailed process provided by the cryo-EM study offers a clear view of these dynamic states, from an inactive state (open AHD and GTP unbound) to an active state (closed AHD and GTP bound) and ultimately to the dissociation of the G protein from the receptor. This research not only deepens our understanding of a key biological process but also provides a valuable map for pharmaceutical researchers to design new therapies. With this high-resolution, dynamic view of GPCR activation, scientists can better tackle diseases by targeting specific steps within the G-protein activation cycle. This highlights the power of advanced imaging techniques like cryo-EM in solving complex biological puzzles and underscores the importance of understanding cellular processes at the molecular level for therapeutic advancements. Beyond GPCRs, the methods and insights from this study can be applied to other fast, transient biological processes. This approach is valuable for understanding the dynamics of other complex signaling pathways and molecular interactions, offering a deeper understanding of cellular processes at the molecular level.
Premont, R. T. & Gainetdinov, R. R. Physiological roles of G protein-coupled receptor kinases and arrestins. Annu Rev Physiol 69, 511-534 (2007).
Papasergi-Scott, M. M. et al. Time-resolved cryo-EM of G-protein activation by a GPCR. Nature (2024).
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