Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity

Abstract

We outline a powerful method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli. For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type. This mutant also shows enhanced heterologous expression in eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) and greater stability when solubilized and purified, indicating that the biophysical properties of the protein had been under the pressure of selection. These improvements arise from multiple small contributions, which would be difficult to assemble by rational design. In a second screen, we rapidly pinpointed a single amino acid substitution in wild type that abolishes antagonist binding while retaining agonist-binding affinity. These approaches may alleviate existing bottlenecks in structural studies of these targets by providing sufficient quantities of stable variants in defined conformational states.

Fig. 1.

General selection scheme for increasing expression level (steps 1, 2, 3a, 4, back to 2) and altering ligand selectivity (steps 1, 2, 3b, 4, back to 2).

Fig. 2.

Percentage radioligand binding to NTR1, D03 (evolved expression variant), and G10 (evolved selectivity variant) with unlabeled agonist (NT) and antagonist (SR 48692) competitors (used at 5 μM each). The radioligand was used at 10 nM. D03 retains the ligand binding properties of NTR1, whereas G10, which was evolved under selection pressure to abolish binding to SR 48692, no longer exhibits high affinity to antagonist.

Fig. 3.

Ca²⁺ signaling in HEK293T cells expressing NTR1, D03, or D03-L167R. Single-cell measurements were performed and the resulting signal was classified as an oscillatory, plateau, or transient response (see Methods, SI Text, and Fig. S11).

Fig. 4.

Comparison of total GPCR yields in P. pastoris by immunoblot analysis. The C-terminal biotinylation was detected with a streptavidin-alkaline phosphatase conjugate. Three independent clones of each construct and a clone with an “empty” vector control (ctrl) integrated into the P. pastoris genome were analyzed (20 μg total membrane protein loaded per lane). Full-length, processed GPCR should run at the position of the arrow. The ≈60 kDa band present in all receptor samples represents the unprocessed precursor form (see Fig. S14). The ≈65 kDa band, which is more pronounced in the D03 samples, also reacts with both M1 and M2 anti-Flag antibodies (data not shown) and likely represents a compact dimeric form. The high molecular weight smear appears to be aggregated receptor, as it is diminished in D03 and D03-L167R. The band at ≈23 kDa, which also appears in the empty vector control, is a P. pastoris protein.

Fig. 5.

Kinetics of thermal denaturation of purified NTR1 (●), D03 (▲), and D03-L167R (■) as fusion proteins with maltose binding protein and thioredoxin. Equal amounts of each receptor were incubated at 45°C and specific activities were measured at various time points by saturation binding assays (see Methods). While NTR1 retains only ≈7% of its specific binding activity after 175 min, the evolved receptors D03 and D03-L167R retain ≈65% and ≈55% of their respective activities after the same interval of time. Results are duplicate measurements from a representative experiment.