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Review
. 2021 Mar 8;26(5):1465.
doi: 10.3390/molecules26051465.

Engineering of Challenging G Protein-Coupled Receptors for Structure Determination and Biophysical Studies

Yann Waltenspühl  1 , Janosch Ehrenmann  1 , Christoph Klenk  1 , Andreas Plückthun  1
Affiliations
Review

Engineering of Challenging G Protein-Coupled Receptors for Structure Determination and Biophysical Studies

Yann Waltenspühl et al. Molecules. .

Abstract

Membrane proteins such as G protein-coupled receptors (GPCRs) exert fundamental biological functions and are involved in a multitude of physiological responses, making these receptors ideal drug targets. Drug discovery programs targeting GPCRs have been greatly facilitated by the emergence of high-resolution structures and the resulting opportunities to identify new chemical entities through structure-based drug design. To enable the determination of high-resolution structures of GPCRs, most receptors have to be engineered to overcome intrinsic hurdles such as their poor stability and low expression levels. In recent years, multiple engineering approaches have been developed to specifically address the technical difficulties of working with GPCRs, which are now beginning to make more challenging receptors accessible to detailed studies. Importantly, successfully engineered GPCRs are not only valuable in X-ray crystallography, but further enable biophysical studies with nuclear magnetic resonance spectroscopy, surface plasmon resonance, native mass spectrometry, and fluorescence anisotropy measurements, all of which are important for the detailed mechanistic understanding, which is the prerequisite for successful drug design. Here, we summarize engineering strategies based on directed evolution to reduce workload and enable biophysical experiments of particularly challenging GPCRs.

Keywords: G protein-coupled receptors; NK1R; NTS1R; PTH1R; directed evolution; protein engineering.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1
Figure 1
Workflow of E. coli-based selection methods. Evolution of GPCRs is initiated by diversification of the receptor gene. The resulting DNA library is used for transformation of E. coli cells, so that each cell takes up at most one plasmid molecule. Subsequently, one receptor variant is expressed in the inner membrane of a bacterial cell. To probe surface expression levels with a fluorescently labelled ligand (red star), the E. coli outer membrane is permeabilized (dashed oval). After incubation with fluorescently labelled ligand to bind to saturation, cell surface expression levels determine the number of bound ligands, and the cells exhibiting the highest fluorescence (i.e., cells with the highest GPCR expression) are enriched by fluorescence-activated cell sorting (FACS). Sorted cells are propagated in growth medium. Re-grown cell pools can either be subjected to additional rounds of selection or plasmids can be isolated for analysis of individual clones.
Figure 2
Figure 2
Workflow of generic selection of GPCRs. Evolution of GPCRs is initiated by diversification of the receptor gene. To enable ligand-independent assessment of surface expression, the receptor is fused N-terminally to a fluorogen-binding protein (cyan bars) and C-terminally to a fluorescent protein (yellow bars). The resulting DNA library is used for transformation of E. coli cells, so that each cell takes up at most one plasmid molecule. Subsequently, one receptor variant is expressed in the inner membrane of a single cell. After incubation with a fluorogen (e.g., malachite green attached to a short polyethylene glycol (PEG) molecule that makes it cell-impermeable, red star), its fluorescence greatly increases and cell surface expression levels determine the number of bound fluorophores, and the cells exhibiting the highest fluorescence (i.e., cells with the highest GPCR expression) are enriched by FACS. Sorted cells were propagated in growth medium. Re-grown cell pools can either be subjected to additional rounds of selection or plasmids can be isolated for analysis of individual clones.
Figure 3
Figure 3
Workflow of Cellular High-Throughout Encapsulation, Solubilization and Screening (CHESS). Evolution of GPCRs is initiated by diversification of the receptor gene. The resulting DNA library is used for transformation of E. coli cells, so that each cell takes up at most one plasmid molecule. Subsequently, one receptor variant is expressed in the inner membrane of a bacterial cell. To probe receptor stability in detergents, the E. coli cells are encapsulated by several polymer layers (black dashed oval) and the cell membranes are solubilized with the detergent of choice. The polymer layers retain the shape of the original cell and convert it into a nanoscopic dialysis bag, as detergent molecules and ligands can traverse the capsule, while proteins cannot. After incubation with the fluorescently labelled ligand (red star), functional receptor levels determine the amount of fluorescent ligand retained. The capsules exhibiting the highest fluorescence (i.e., capsules containing the most stable GPCRs) are enriched by FACS. Enriched capsules of course cannot be re-grown, therefore the sequences of GPCR contained in the enriched capsules are amplified by polymerase chain reaction (PCR) and cloned into a new plasmid backbone to repeat the process or analyze individual clones.
Figure 4
Figure 4
Workflow of Saccharomyces cerevisiae-Based Receptor Evolution (SaBRE). Evolution of GPCRs is initiated by diversification of the receptor gene. The resulting DNA library is used for transformation of S. cerevisiae cells, so that each cell takes up at most one plasmid molecule. Subsequently, one receptor variant is expressed in the plasma membrane of a single cell. To probe surface expression levels with a fluorescently labelled ligand (red star), the yeast cell wall was permeabilized (dashed oval). After incubation with fluorescently labelled ligand to bind to saturation, cell surface expression levels determine the number of bound ligands, and the cells exhibiting the highest fluorescence (i.e., cells with the highest GPCR expression) are enriched by FACS. Sorted cells were propagated in growth medium. Re-grown cell pools can either be subjected to additional rounds of selection or plasmids can be isolated for analysis of individual clones.
Figure 5
Figure 5
Engineered receptors are stabilized in biologically relevant states. Conformational changes during class A receptor activation. (a) Superposition of NTS1R-HTGH4 (PDB ID: 4BWB), NTS1R-EL (PDB ID: 5T04), and wtNTS1R:Gαi (PDB ID: 6OS9), all in complex with NT 8–13. Transmembrane helices V and VI are highlighted in color. Conformational changes are indicated by black arrows. (b) Schematic overview of receptor conformational states. From left: inactive conformation with expanded extracellular binding pocket and contracted intracellular helix bundle (blue, none of the structures above correspond to this state). Intermediate conformation with extracellular active-like contracted binding site, but only loosely expanded intracellular bundle (yellow helix VI, corresponding to the yellow and orange structures above). The mobility of the cytoplasmic side is indicated by orange lines. Fully active state, indicated by contracted binding pocket and outward movement of intracellular helix ends of helices V and VI (magenta).
Figure 6
Figure 6
Engineered receptors allow detailed insights into GPCR ligand interactions. Conformational changes explaining insurmountable antagonism of aprepitant. Superposition of NK1R:aprepitant (PDB ID: 6HLO) and NK1R:CP-99,994 (PDB ID: 6HLL). For clarification, black arrows indicate sidechain movements.
Figure 7
Figure 7
Crystal structure of engineered PTH1R is stabilized in an intermediate state. Conformational changes during class B receptor activation. (a) Superposition of GCGR (PDB ID: 5YQZ), PTH1R (PDB ID: 6FJ3), and wtPTH1βR:Gαs (PDB ID: 6NBF). Transmembrane helices I, VI, and VII are highlighted in color. Conformational changes are indicated by black arrows. (b) Schematic overview of receptor conformational states. From left: inactive conformation with contracted intracellular and extracellular helix portions. Intermediate conformation with extracellular active-like expanded binding site, but only loosely expanded intracellular helix bundle (indicated by orange lines). Fully active state, indicated by expanded binding pocket and outward movement of intracellular ends of helix VI.
Figure 8
Figure 8
Applications of engineered receptors outside crystallography. Possible applications of receptor variants engineered for stability and enhanced functional expression. Thermostabilized receptors are well suited for methods requiring long half-life in detergent such as surface plasmon resonance (SPR) or nuclear magnetic resonance (NMR). Schematically, mutations introduced are indicated by red and purple stars. Purification tag fused to the receptors is depicted in light blue.
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