Dataset on Galanin Receptor 3 mutants that improve recombinant receptor expression and stability in an agonist and antagonist bound form

Galanin Receptor 3 (GALR3) is a G-protein-coupled receptor with a widespread distribution in the brain and plays a role in a variety of physiologic processes including cognition/memory, sensory/pain processing, hormone secretion, and feeding behavior. Therefore, GALR3 is considered an attractive CNS drug target (Freimann et al., 2015) [1]. This dataset contains GALR3 point mutants that improve recombinant protein expression and thermal stability of the receptor contained in virus-like particles (VLPs) or obtained by detergent-purification of baculovirus-infected insect cells. The mutations listed can be grouped in those that improve the stability of the agonist-bound and the antagonist-bound form of the receptor. Protein characteristics in terms of protein expression and thermal stability were comparable between GPCR-VLP and GPCR overexpressing Sf9 cultures. The further analysis and detailed results of these mutants as well as their impact on biophysical assay development for drug discovery can be found in “Method for Rapid Optimization of Recombinant GPCR Protein Expression and Stability using Virus-Like Particles” (Ho et al., 2017) [2].


Value of the data
The GALR3 mutants listed here aid in increasing the recombinant protein expression yield and stabilization of the receptor in either the agonist or antagonist-bound form.
Mutational analysis of the GALR3 variants was performed in VLPs. The chemically stabilizing environment of the phospholipid bilayer in a VLP eliminates the need for recombinant overexpression, purification and detergent solubilization during the iterative protein engineering process.
GALR3 protein quality from VLPs was comparable to detergent-purified receptors from overexpressing Sf9 cultures.

Data
The dataset of this article provides information on GALR3 mutants that stabilize the receptor in either an agonist-bound or antagonist-bound form. Table 1 shows how 23 of the 210 point mutants expressed on VLPs increase [ 125 I]-galanin binding and thermal stability compared to WT. In addition, a direct correlation of the B max values with recombinant expression yields of the mutants in Sf9 cultures was shown (Table 1 and Fig. 1). Combinations of these mutants were made to find the best thermostabilizing GALR3 agonist-bound (Table 2) and GALR3 antagonist-bound (Table 3) variants.

Characteristics of GALR3 point mutants in VLP versus detergent purified receptors from overexpressing Sf9 cultures
Initial rank ordering of 210 GALR3-VLP point mutants, mostly located in GALR3 transmembrane helices, was based on expression yield obtained from a radiometric assay measuring the binding of    (Table 1). 19 GALR3 point mutants were then recombinantly expressed in Sf9 insect cells to measure protein expression yields as determined by SDS-PAGE/ Western blot band intensities (Fig. 1). Expression levels of the 19 single mutants followed the same rank-order as the B max values measured by the radiometric assay on the corresponding VLP samples ( Fig. 1 and Table 1). Mutants are shown for which Z 50% monodispersity could be achieved after mid-scale (300 ml) recombinant expression in insect cells followed by detergent solubilization and purification. Expression was done once and provided enough material for one Tm measurement. Receptor purity and monodispersity was analyzed using SDS-PAGE and analytical size-exclusion chromatography (SEC). Thermal stability was measured by the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3coumarinyl)phenyl]maleimide (CPM) assay [3] *Note that WT GALR3 protein was mostly aggregated and its Tm value cannot be accurately measured. However, GALR3WT was added to the table as a reference. Mutants are shown for which Z 50% monodispersity could be achieved after mid-scale (300 ml) recombinant expression in insect cells followed by detergent solubilization and purification in the presence of antagonist DNS001131702 or DNS001355175. Expression was done once and provided enough material for one Tm measurement. Thermal stability was measured by the CPM assay [3].

GALR3 agonist-bound thermostabilizing variants
The best GALR3 single point mutants were combined for mid-scale (300 ml) recombinant expression in Sf9 insect cells and protein parameters such as expression yield, monodispersity and thermal stability in the presence of galanin are listed in Table 2, summarizing the best mutant combinations that improved the agonist-bound protein characteristics of the receptor.

GALR3 antagonist-bound thermostabilizing variants
In order to find the stabilizing GALR3 antagonist-bound mutants, we used the data from the GALR3-VLP [ 125 I]-galanin binding assay described above to select 88 single point mutants with B max values Z80% of WT, assuming that these displayed enough receptor density to be further evaluated in a size-exclusion chromatography/liquid chromatography mass spectroscopy (SEC/LC-MS) based binding assay. Prior to SEC/LC-MS, the GALR3-VLP samples were incubated with GALR3 antagonist, DNS000135175 (Ki ¼55 nM) or DNS001131702 (Ki¼127 nM) and subjected to a temperature gradient ranging from 15°C to 55°C. Before the acquisition of the LC-MS spectra of the ligand, unbound ligand was removed by SEC. A total of 5 single point mutants were found to stabilize the antagonist form of GALR3 by Z4°C when compared to the WT form, and rank-ordered from most to least stabilizing as: A223L (ΔT ¼8.3°C), A218L (ΔT ¼7.2°C), R120A (ΔT ¼5.4°C), V116A (ΔT ¼4.6°C), E224A (ΔT ¼4.2°C).
Similar to the GALR3 agonist-bound studies, the best GALR3 single point mutants were combined for recombinant expression in Sf9 insect cells and protein parameters such as expression yield, monodispersity and thermal stability in the presence of a GALR3 antagonist-bound form are listed in Table 3.