Generating thermostabilized agonist-bound GPR40/FFAR1 using virus-like particles and a label-free binding assay

Figures & data

Figure 1. Snake plot of the amino acid sequence of human GPR40 showing the thermostabilized sites. Blue, increase (%) in activity in the fasiglifam-binding condition (G29, T31, L38, C127, A128, A129, W131, A132, L133, V134, E145, G149 and V225); yellow, increase (%) in activity in the compound-free condition (V64, A109, Y122, L135, A199, F200, G204, V237, G265, T286 and V287); red, increase (%) in activity in both conditions (L42, F88, G103, R104, Y202 and K285); gray, not tested or fasiglifam binding signal for the mutant <25% of the signal for the wild-type; *binding signal of fasiglifam not determined for both tyrosine-to-alanine and tyrosine-to-phenylalanine mutants; **binding signal of fasiglifam not determined for tyrosine-to-alanine mutant but determined for tyrosine-to-phenylalanine mutant; ***increase (%) in activity for tyrosine-to-phenylalanine mutant; ****increase (%) in activity for both tyrosine-to-alanine and tyrosine-to-phenylalanine mutants.

Figure 2. Apparent T_m (, °C) of the wild-type and the thermostabilized four-point mutant of GPR40. Binding signals at each temperature were normalized as percentages of the binding signal at 4 °C. This experiment was performed in sextuplet. Data are expressed in means ± SEM and were fitted using the sigmoidal dose-response curve equation of Prism 5.03 software. Closed circles, the wild-type under format A condition; open circles the wild-type under format B condition; closed triangles, thermostabilized four-point mutant under format A condition; open triangles, thermostabilized four-point mutant under format B condition.

Table 1. Apparent T_m (, °C) values of wild-type and single-point thermostabilized mutants of GPR40.

	(Format A)	(Format A)	(Format B)	(Format B)
WT	37.2	–	35.5	–
T31A	38.3	1.1	38.3	2.8
L42A	42.1	4.9	39.8	4.3
V64A	39.6	2.4	37.7	2.3
F88A	44.0	6.8	43.9	8.3
G103A	42.7	5.5	41.2	5.7
R104A	40.9	3.7	39.2	3.7
Y122F	41.1	3.8	39.2	3.7
E145A	42.8	5.6	39.6	4.1
A199V	39.8	2.6	37.6	2.1
Y202A	40.5	3.3	38.9	3.4
Y202F	43.7	6.5	42.7	7.2
V225A	40.4	3.2	38.2	2.7
G265A	41.1	3.9	38.9	3.3

Table 2. values (°C) of wild-type and the thermostabilized four-point mutant of GPR40.

	(°C)
	Format A	Format B
Wild-type	38.3 ± 0.3	36.5 ± 0.3
L42A/F88A/G103A/Y202F	55.7 ± 0.3	55.7 ± 0.4

Each data point was assayed in sextuplet means ± SEM

Figure 3. Fasiglifam binding affinity for the wild-type and the thermostabilized four-point mutant of GPR40. Saturation binding curves of fasiglifam for the wild-type (a) and the thermostabilized four-point mutant (b) were determined by the SEC/LC-MS-based binding assay. The specific binding signal for the receptor (open triangles) was calculated as the difference between the total binding signal (solid circles) and the non-specific binding signal (solid squares). This experiment was performed in sextuplet Data are expressed in means ± SEM and were fitted using the one site-specific binding curve equation of Prism 5.03 software.

Table 3. values of fasiglifam and B_max for wild-type and the thermostabilized four-point mutant of GPR40.

Sample form	Construct	(nM)	Bmax (fmol)	Total protein per assay (ng)
VLP	Wild-type*	11.9 ± 1.4	3.6 ± 0.1	75
	L42A/F88A/G103A/Y202F*	11.4 ± 3.1	2.8 ± 0.2	75
	FLAG/wild-type†	8.0 ± 0.8	3.6 ± 0.1	75
	FLAG-L42A/F88A/G103A/Y202F†	11.8 ± 1.2	1.7 ± 0.04	75
Cytoplasmic membrane	Wild-type†	7.7 ± 0.7	2.7 ± 0.1	1250
	L42A/F88A/G103A/Y202F†	8.3 ± 0.6	3.6 ± 0.1	1250

*Each data point was assayed in sextuplet means ± SEM

†Each data point was assayed in quadruplicate means ± SEM

Figure 4. Agonistic activity of fasiglifam (a) and DHA (b) in wild-type (circles) and the thermostabilized four-point mutant of GPR40 (squares). The experiment was performed in quadruplicate. Data are expressed in means ± SEM and were fitted using the dose-response equation of Prism 5.03 software.

Figure 5. Transient expression level of wild-type and the thermostabilized four-point mutant GPR40 on the HEK-293T cell surface detected by FACS. The experiment was performed in triplicate. Data are expressed in means ± SEM.

Table 4. Agonistic activity (EC₅₀) of fasiglifam and DHA for the FLAG-tagged wild-type and FLAG-tagged thermostabilized four-point mutant of GPR40.

	log EC50 (M)
	Fasiglifam	DHA
FLAG/wild-type	−7.89 ± 0.11	−6.32 ± 0.08
FLAG/L42A/F88A/G103A/Y202F	−4.69 ± 0.05	−4.70 ± 0.02

Each data point was assayed in quadruplicate means ± SEM