G protein-coupled receptors (GPCRs), as important drug targets, have always faced a key challenge in drug screening, such as the screening of nanobodies targeting extracellular regions. A recently published study proposes an innovative method to stabilize GPCRs in a specific conformation, providing a new approach for nanobody screening.
Traditional GPCR purification and screening are limited by their conformational instability, particularly in capturing their active state. Although molecules such as agonists can help stabilize the active conformation, they often occupy the binding pocket, hindering the screening of new ligands. This study developed a universal conformational stabilization strategy: by designing a fusion protein de novo and linking TM5 and TM6, TM6 is locked in either an "inward" (inactive) or "outward" (active) conformation, thereby achieving stable control over specific states of the GPCR.
Fig 1. Schematic diagram of stable M1R conformation design [1]
A. Dynamic native GPCRs are stabilized in inactive or active conformations through protein design.
B. A fusion protein containing the α5 helix of the G protein is embedded between TM5 and TM6 to stabilize the active conformation of the GPCR.
Using the M1 muscarinic acetylcholine receptor (M1R) as a model, the research team established a protein design flow that integrates computation and experimentation:
Taking M1R-G11CT as an example, its high-resolution structure serves as the "design blueprint."
The G protein α5 helix (G11CT) is identified as a key module driving and stabilizing the conformation; suitable residues on TM5 and TM6 are selected as fusion sites.
RFdiffusion is used to generate the backbone of the fusion protein between TM5 and TM6, forming a binding pocket adapted to G11CT.
ProteinMPNN is used to design the optimal amino acid sequence for the generated backbone.
AlphaFold2 is used for structure prediction, followed by pLDDT scoring to screen high-confidence designs, which are then synthesized and validated.
Fig 2. Design and biochemical characterization of M1R-G11CT [1]
C. Overview of the design process of the M1R-G11CT fusion protein.
D. Molecular sieve chromatography (SEC) curve and corresponding SDS-PAGE analysis of M1R-G11CT, showing that the protein is well folded and homogeneous, requiring no additional stabilizing ligands.
E. Comparison of M1R-G11CT and M1R-ΔICL3 in competitive binding experiments with iperoxo. This shows that M1R-G11CT has enhanced agonist affinity and maintains an exceptionally stable active state.
F. Comparison of M1R-G11CT and M1R-miniGsq in competitive binding experiments with iperoxo. This shows that M1R-G11CT has homogeneity and stability.
The intracellular region of GPCRs undergoes significant conformational changes and has a large surface area upon activation. During screening, antibodies targeting the intracellular region are preferentially enriched, while antibodies specific to the extracellular region are typically at a disadvantage and excluded. The research team proposed an "intracellular orthogonal screening" strategy: utilizing receptor pairs with similar extracellular structures but different intracellular fusion proteins, including stable active M1R and inactive M1R, multiple rounds of magnetically activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) were used, with the receptor concentration gradually decreasing, to screen a synthetic yeast-displayed nanobody library, successfully identifying conformation-specific nanobodies targeting the extracellular region.
Fig 3. Intracellular orthogonal screening of extracellular nanobodies [1]
A. Schematic diagram of the screening strategy based on conformationally stable GPCR.
B. Schematic diagram of the intracellular orthogonal screening method for separating inactive state (M1R-ΔICL3 and M1R-Clip1) selective nanobodies.
C. Schematic diagram of the intracellular orthogonal screening method for separating active state (M1R-G11CT and M1R-G11CTb) selective nanobodies.
D. Yeast surface staining analysis to verify the conformational selectivity of nanobodies, demonstrating their ability to distinguish receptor states.
E-G. Two-dimensional class average plots of cell-based functional experiments and inactive state specific nanobodies (NbF7, NbF3 and NbA12) show that these nanobodies are located in extracellular binding.
● Methodological Innovation:
It provides a universal "conformation-locked" platform, overcoming the limitations of dynamic receptors in structural analysis and screening applications.
● Drug Development Empowerment:
It offers a new tool for developing highly selective therapeutics, significantly improving the efficiency and specificity of drug screening, and opening new avenues for GPCR-targeted drug development.
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References: [1] Zhang X, Gao KX, Nie J, et al. Extracellular nanobody screening using conformationally stable GPCR variants. Proc Natl Acad Sci U S A. 2025 Nov 4, 122(45):e2508879122. doi: 10.1073/pnas.2508879122.
Download link for the original reference: Extracellular Nanobody Screening using Conformationally Stable GPCR.pdf
