In a groundbreaking advance that addresses one of the most intricate challenges in cellular signaling, researchers have unveiled a novel approach to selectively modulate G protein-coupled receptor (GPCR) activity through the sophisticated design of allosteric modulators. This cutting-edge study unravels the enigmatic mechanisms by which a small molecule, SBI-553, achieves subtype-selective antagonism of G proteins, offering fresh insights into receptor-transducer specificity that could transform drug discovery paradigms targeting GPCRs.
GPCRs stand as paramount signaling conduits in eukaryotic cells, orchestrating myriad physiological responses by coupling to heterotrimeric G proteins. These G proteins, which vary widely in subtype, interact with GPCRs predominantly via their highly divergent C-terminal regions of the α-subunit. Traditionally, the recruitment and activation of different G proteins by a single GPCR was considered a somewhat uniform process. However, subtle determinants that govern selective engagement of specific G protein subtypes have remained elusive.
The recent investigation pivots around SBI-553, an allosteric molecule previously shown to inhibit Gq/11 protein activation mediated by the neurotensin receptor 1 (NTSR1). The essential question addressed was whether this antagonism stemmed solely from indirect mechanisms such as β-arrestin recruitment or if direct obstruction of G protein binding sites occurred. By engineering chimeric G protein constructs that swapped the critical C-terminal residues between Gq and GoA proteins, the study decisively demonstrated that the primary structure of the G protein C terminus dictates the effectiveness of SBI-553-mediated antagonism.
Specifically, when the C-terminal five amino acids of GoA were replaced with those of Gq, the resulting chimera became susceptible to inhibition by SBI-553, despite native GoA being fully permissive to G protein activation in SBI-553’s presence. Conversely, introducing GoA’s C-terminal residues into Gq attenuated the antagonist’s potency, underpinning the indispensable role of these residues in determining receptor-G protein specificity. By extending this approach further and exchanging the terminal 13 residues in Gq with those from GoA, the construct exhibited near-complete insensitivity to SBI-553 across all but the highest tested concentrations, emphasizing cumulative effects rather than single residues driving this specificity.
Intriguingly, point mutations targeting four unique residues within the Gq C terminus, hypothesized to mediate SBI-553 sensitivity, fell short of recapitulating the full C-terminal swap’s effects. Each single substitution—be it V359Y, N357G, Y356C, or L351N—allowed neurotensin-induced activation to proceed unaffected, with SBI-553 maintaining its full antagonistic capacity. This finding underscores that the allosteric modulation and selectivity are not directed by individual residues but by the holistic conformational dynamics enabled by the entire C-terminal segment.
Delving deeper into the structural underpinnings, the researchers leveraged high-resolution cryo-electron microscopy structures of NTSR1 bound to neurotensin and the respective mini-G proteins. Absent SBI-553, both GoA and Gq maintain a ‘closed’ α-helical conformation within the receptor intracellular core, overlapping extensively in space. However, when modeling the binding of SBI-553 onto these complexes, steric clashes emerge, implying that coexistence demands conformational rearrangements.
Notably, in the presence of SBI-553, GoA undergoes a striking transformation to an ‘open’ conformation. Here, the Gα C-terminal helix tilts approximately 14 degrees towards transmembrane helix 1 and unwinds partially over the last five residues. This repositioning retracts the α-helix about 4 angstroms shallower into the receptor and creates van der Waals contacts with SBI-553’s molecular moieties. The uniqueness of this conformation stands out, as comprehensive superpositions of known GPCR-G protein complexes fail to reveal such alternative binding modes, suggesting SBI-553 itself induces this rare structural state.
This alternative binding paradigm can explicate why SBI-553 selectively inhibits Gq/11 proteins: the energetic feasibility of adapting this shallow-binding ‘open’ conformation varies among G protein subtypes. Using in silico homology modeling, the team examined multiple G proteins, including Gq, G11, GoB, Gi variants, and G12/13, by substituting their C-terminal residues onto the GoA ‘open’ conformation template. The models revealed that certain residues in Gq/11 impose energetic penalties—such as needing to adopt disfavored backbone dihedral angles—that impede adopting the open conformation compatible with SBI-553 binding. Conversely, G12 and G13, despite slight energetic concessions, gain stabilizing van der Waals interactions with SBI-553, potentially explaining their differential sensitivities.
Beyond the direct structural compatibility, the functional consequences of SBI-553 extend to receptor activation dynamics. The compound interacts with the crucial E/DRY motif of NTSR1, specifically targeting the arginine residue, thereby disrupting the receptor’s inactive conformation and facilitating an active-like state. Concurrently, the induced ‘open’ G protein conformation resembles intermediate states along the G protein activation pathway, indicating that SBI-553 modulates receptor and transducer activation states in a coordinated manner rather than purely blocking their association.
Attempts to solve the empirical structure of an NTSR1–Gq–SBI-553 complex have thus far been unsuccessful, likely due to the biochemical incompatibility stemming from Gq’s reluctance or inability to adopt the necessary open conformation occluded by SBI-553. This aligns with the observed pharmacological profile where SBI-553 antagonizes Gq but not GoA in cellular assays.
These findings herald a conceptual leap in understanding how allosteric modulators can reprogram GPCR signaling specificity. Instead of acting merely as inhibitors or activators, such compounds can sculpt the conformational landscape of receptor-transducer interfaces, selectively stabilizing or destabilizing specific protein-protein interactions based on subtle structural features. This nuanced mechanism affords unprecedented finesse in therapeutic targeting by enabling pathway-selective modulation, potentially minimizing adverse off-target effects attributed to promiscuous G protein activation.
Moreover, the study synergizes structural biology, protein engineering, and computational modeling to elucidate the molecular grammar governing receptor-G protein coupling specificity. The profound implications extend to numerous GPCR systems, given the conserved architecture yet diverse functional outcomes mediated via distinct G protein subtypes. By exploiting the inherent conformational plasticity of Gα subunits and the receptor intracellular crevice, bespoke allosteric ligands like SBI-553 can be rationally designed to tailor signaling cascades.
In the broader context of pharmacology, this work underscores the value of examining receptor-transducer interactions beyond traditional agonism and antagonism frameworks. Allosteric modulators that influence protein conformational ensembles may emerge as a predominant class of modulators that achieve selective signaling bias in complex physiological environments. This approach holds promise for conditions where specific G protein pathways contribute to pathological states, such as neurological disorders, cardiovascular diseases, and cancer.
In conclusion, the pioneering elucidation of SBI-553’s mode of action illustrates how fine structural determinants within the G protein C terminus govern the allosteric modulation landscape at GPCRs. The insights gleaned define new avenues for precision drug design, empowering the development of selective signaling modulators that harness the nuanced conformational dynamics between receptors and their diverse transducer partners.
Subject of Research:
The study focuses on the molecular determinants of G protein subtype selectivity in GPCR signaling, specifically examining how an allosteric modulator (SBI-553) influences the coupling preferences of the neurotensin receptor 1 (NTSR1) towards Gq/11 versus GoA proteins by altering G protein C-terminal conformation.
Article Title:
Designing allosteric modulators to change GPCR G protein subtype selectivity
Article References:
Moore, M.N., Person, K.L., Robleto, V.L. et al. Designing allosteric modulators to change GPCR G protein subtype selectivity. Nature (2025). https://doi.org/10.1038/s41586-025-09643-2
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