Expression, purification, crystallization, and preliminary X-ray crystallographic studies of the human adiponectin receptors, AdipoR1 and AdipoR2

Abstract

The adiponectin receptors (AdipoR1 and AdipoR2) are membrane proteins with seven transmembrane helices. These receptors regulate glucose and fatty acid metabolism, thereby ameliorating type 2 diabetes. The full-length human AdipoR1 and a series of N-terminally truncated mutants of human AdipoR1 and AdipoR2 were expressed in insect cells. In small-scale size exclusion chromatography, the truncated mutants AdipoR1Δ88 (residues 89-375) and AdipoR2Δ99 (residues 100-386) eluted mostly in the intact monodisperse state, while the others eluted primarily as aggregates. However, gel filtration chromatography of the large-scale preparation of the tag-affinity-purified AdipoR1Δ88 revealed the presence of an excessive amount of the aggregated state over the intact state. Since aggregation due to contaminating nucleic acids may have occurred during the sample concentration step, anion-exchange column chromatography was performed immediately after affinity chromatography, to separate the intact AdipoR1Δ88 from the aggregating species. The separated intact AdipoR1Δ88 did not undergo further aggregation, and was successfully purified to homogeneity by gel filtration chromatography. The purified AdipoR1Δ88 and AdipoR2Δ99 proteins were characterized by thermostability assays with 7-diethylamino-3-(4-maleimidophenyl)-4-methyl coumarin, thin layer chromatography of bound lipids, and surface plasmon resonance analysis of ligand binding, demonstrating their structural integrities. The AdipoR1Δ88 and AdipoR2Δ99 proteins were crystallized with the anti-AdipoR1 monoclonal antibody Fv fragment, by the lipidic mesophase method. X-ray diffraction data sets were obtained at resolutions of 2.8 and 2.4 Å, respectively.

Fig. 1

Purification of full-length AdipoR1. a Gel filtration chromatogram of the full-length AdipoR1 expressed in High Five insect cells. The proteins were purified by FLAG-affinity chromatography, and then loaded onto a HiLoad 16/600 Superdex 200 column. The main peak retention volume is labeled in black (46.7 ml). The absorbances at 280 and 254 nm are colored blue and red, respectively. b SDS-PAGE analysis of the gel-filtration chromatographic fractions containing the full-length AdipoR1. Fractions indicated by the orange and green lines along the horizontal axis in a were analyzed by SDS-PAGE. Lane F, the eluate from FLAG-affinity chromatography; Lane M, molecular-weight markers (kDa)

Fig. 2

Screening of the N-terminally truncated mutants of AdipoR1 and AdipoR2. Several N-terminal deletion mutants were analyzed by size-exclusion chromatography. a Schematic representations of human AdipoR1 (top) and AdipoR2 (bottom). b Amino acid sequences of the N-terminal regions of human AdipoR1 and AdipoR2. The starting amino acid residues of the N-terminally truncated mutants of AdipoR1 and AdipoR2 are numbered in red. Amino acids in putative transmembrane domains are shown in gray letters. The sequences were aligned with ClustalW [38]. c Size-exclusion chromatograms of the full-length form and the N-terminally truncated mutants of AdipoR1. d Detection of the AdipoR1 protein in the size-exclusion chromatography fractions by western blotting with the anti-FLAG M2 antibody. e Size-exclusion chromatogram of AdipoR2Δ99

Fig. 3

Large-scale preparation of FLAG-tagged AdipoR1Δ88 and AdipoR2Δ99 expressed in High Five cells. In the chromatograms (a, c–h, j, k, m, n), the absorbances at 280 and 254 nm, and the NaCl concentration are shown in blue, red, and light green, respectively. a Gel filtration chromatogram of FLAG-tagged AdipoR1Δ88 expressed in High Five insect cells. The proteins were purified by FLAG-affinity chromatography, and then chromatographed on a HiLoad 16/600 Superdex 200 column. The main peak elution volume is labeled in black (58.8 ml). b SDS-PAGE analysis of the gel-filtration chromatographic fractions containing the FLAG-tagged AdipoR1Δ88. Fractions indicated by the orange and green lines along the horizontal axis in a were analyzed by SDS-PAGE. Lane M, molecular-weight markers (kDa). c Anion-exchange chromatogram of the FLAG-tagged AdipoR1Δ88, with isocratic elution by 100 mM NaCl in buffer A, and subsequently with gradient elution by 100–1,000 mM NaCl in buffer A. d, e Gel filtration chromatograms of the peak 1 (d) and peak 2 (e) fractions, indicated by the orange and green lines in c, respectively. f Anion-exchange chromatogram of the FLAG-tagged AdipoR1Δ88, with isocratic elution by 200 mM NaCl in buffer A, and subsequently with gradient elution by 200–1,000 mM NaCl in buffer A. g, h Gel filtration chromatograms of the flow-through (g) and adsorbed (h) fractions, indicated by the orange and green lines in f, respectively. i SDS-PAGE analysis of the FLAG-tagged AdipoR1Δ88. Lane M, molecular-weight markers (kDa); lane 1, the membrane fraction; lane 2, DDM-solubilized membrane proteins in the supernatant after ultracentrifugation; lane 3, the flow-through fraction from FLAG-affinity chromatography; lane 4, the eluate from FLAG-affinity chromatography; lane 5, the flow-through fraction from anion-exchange chromatography (f); lanes 6–9, the peak fractions of the FLAG-tagged AdipoR1Δ88 from gel filtration chromatography (g). j Anion-exchange chromatogram of AdipoR2Δ99, with isocratic elution by 200 mM NaCl in buffer A and subsequently with gradient elution by 200–1,000 mM NaCl in buffer A. k Gel filtration chromatogram of the flow-through fractions indicated by the orange line in j. l SDS-PAGE analysis of AdipoR2Δ99. Lane M, molecular-weight markers (kDa); lane 1, the membrane fraction; lane 2, DDM-solubilized membrane proteins in the ultracentrifugation supernatant; lane 3, the flow-through fraction from FLAG-affinity chromatography; lane 4, the eluate from FLAG-affinity chromatography; lane 5, the flow-through fraction from anion-exchange chromatography after the TEV protease digestion; lane 6, the flow-through fraction from TALON chromatography; lanes 7–11, the peak fractions of AdipoR2Δ99 from gel filtration chromatography. m, n Gel filtration chromatographic analysis of the purified FLAG-tagged AdipoR1Δ88 (m) and the purified AdipoR2Δ99 (n)

Fig. 4

Characterization of the purified AdipoR1Δ88 and AdipoR2Δ99 proteins. a, b CPM assay of AdipoR1Δ88 (a) and AdipoR2Δ99 (b). c–e TLC analysis of AdipoR1Δ88 (Δ88), the full-length AdipoR1 (FL1), AdipoR2Δ99 (Δ99), and the full-length AdipoR2 (FL2). The lipids were visualized with acetic acid/sulfuric acid [1:1 (v/v)] (c), the phosphomolybdic reagent (d), and the ninhydrin reagent (e). Lane M, polar lipid mixture (Matreya)

Fig. 5

Screening of anti-AdipoR1 antibodies. a Representative ELISA plate results. Yellow areas represent ELISA positive clones. Well C7, enclosed in a red box, indicates clone #43. b FSEC analyses of IgG#43 (top) and IgG#55 (bottom). Peaks p1, p2, and p3 represent the peak of the ternary complex (AdipoR1Δ88, the antibody from hybridoma cells, and the fluorescein–Fab fragment), the antibody complex (the antibody from hybridoma cells and the fluorescein–Fab fragment), and the free fluorescein–Fab fragment, respectively. c SDS-denatured dot blot screening of anti-AdipoR1 antibodies. IgG#17, IgG#21, and IgG#55 stained denatured AdipoR1. IgG#43 and IgG#55 are numbered in red

Fig. 6

Isolation of the AdipoR1Δ88·Fv43 and AdipoR2∆99·Fv43 complexes. a, b Gel filtration chromatograms of the AdipoR1Δ88·Fv43 (a) and AdipoR2∆99·Fv43 (b) complexes. c, d SDS-PAGE analysis of the crystallization samples of the AdipoR1Δ88·Fv43 (c) and AdipoR2∆99·Fv43 (d) complexes

Fig. 7

Crystals of the FLAG-tagged AdipoR1Δ88·Fv43 and AdipoR2∆99·Fv43 complexes. a, b Crystals of the FLAG-tagged AdipoR1Δ88·Fv43 complex. c, d Crystals of the AdipoR2∆99·Fv43 complex. Crystals are shown in bright field (a, c) and under crossed polarizers (b, d)

Fig. 8

X-ray diffraction images of the AdipoR1Δ88·Fv43 complex (a) and the AdipoR2∆99·Fv43 complex (b)