Specificity of receptor-ligand interactions and their effect on dimerisation as observed by electrospray mass spectrometry: bile acids form stable adducts to the RXRalpha

Abstract

Electrospray (ES) mass spectrometry data is presented showing that agonist binding to the nuclear receptor (NR), retinoid X receptor alpha (RXRalpha), is competitive. The competitive nature of agonist binding can be used to discriminate between the specific and non-specific binding of small lipophilic molecules to NRs. Further, data is presented which show that high-affinity ligand binding to the RXRalpha ligand-binding domain (LBD) stabilises the domain homodimer. The results indicate that homodimerisation, a functional property of the receptor associated with the binding of agonist ligands, could be used to discriminate between specific and non-specific binding events. Additionally, we report on the remarkable stability of the gas-phase complex between the RXRalpha LBD protein and endogenous bile acids. Protein-bile acid interactions in the gas phase were found to be surprisingly strong, withstanding 'in-source' fragmentation in the ES interface, and, in the case of taurocholic acid (TCA) and lithocholic acid-3-sulphate (LCA-3-sulphate), collision-induced dissociation within the collision cell of a tandem mass spectrometer. Bile acids were found to be inactive towards RXRalpha in transfection assays, and have not been reported to be ligands for the RXRalpha, although lithocholic acid (LCA) has been found to be a competitor in the photoaffinity labelling of RXRbeta with 9-cis-retinoic acid (9-cis-RA). The observation of strong RXRalpha-bile acid non-covalent complexes in ES mass spectrometry highlight the danger of extrapolating gas-phase binding data to the solution phase and further to a possible biological activity, particularly when surface-active compounds such as bile acids are involved. The introduction of a competitive ligand-binding experiment can alleviate this problem and allow the differentiation between specific and non-specific binding.

Scheme 1

Structures of compounds analysed for RXRα binding by ES mass spectrometry, (A) cholic acid, (B) taurocholic acid, (C) chenodeoxycholic acid, (D) lithocholic acid, (E) lithocholic acid-3-sulphate, (F) LG268, (G) docosahexaenoic acid and (H) dodecylsulphate.

Figure 1

Titrations of RXR agonists and bile acids on transfected cells. High affinity LG268 (solid square) induces RXR activation in the nM range. The fatty acids DHA (C22:6, solid wedge) and DTA (C22:4 solid triangle) show EC₅₀-values in the μM range, whereas the bile acids TCA (solid diamond), CDCA (solid circle) and CA (open square) are all inactive. Note that the bile acid data points overlap on to the x-axis.

Figure 2

Analysis of RXRα under non-denaturing conditions in the absence and presence of agonist ligand. (A) ES mass spectrum of a 10 μM solution of RXRα LBD in 10 mM ammonium acetate, pH 8.1, covering the m/z range 1000 – 4500. Shown in B – E is the most intense charge state of monomeric RXRα ([M+12H]¹²⁺) in the abscence of ligand (B); or in the presence of 25 μM LG268 (C); DHA (D); or 17β-estradiol (E). The +178 Da modification (His₆-tag gluconoylation) is indicated by a filled circle. The spectra were recorded with a sample flow rate of 5 - 10 μL/min, a cone-voltage of 50 V and a collision voltage of 4.2 V, with a source block temperature 80 - 120 °C.

Figure 3

Agonist binding to RXRα is competitive. ES mass spectra showing the 12+ monomer charge state for RXRα in the absence of ligand (A); in the presence of 10 μM DHA (B); and in the presence of 10 μM DHA and LG268 (C). The receptor concentration was 10 μM. The +178 Da modification (His₆-tag gluconoylation) is indicated by a filled circle. The sample flow-rate was 5 μL/min, and data was acquired at a cone voltage of 50 V, a collision voltage of 4.2 V with a source block temperature of 80 °C.

Figure 4

Bile acid and LG268 binding to RXRα is non-competitive. ES mass spectra showing the RXRα monomer 12+ charge state (receptor concentration 10 μM) in the absence of ligand (A); in the presence of 10 μM LG268 (B); or in the presence of 10 μM LG268 and 30 μM TCA (C and D). The +178 Da modification (His₆-tag gluconoylation) is indicated by a filled circle and bound bile acid molecules by a filled diamond. Sample flow rate was 5 μL/min, in (A), (B) and (C) the cone-voltage was 50 V, while in (D) it was 195 V. In all spectra the collision voltage was 4.2 V with a source block temperature of 80 °C.

Figure 5

Bile acid and fatty acid binding to RXRα is non-competitive. Spectra of the RXRα monomer 12+ peak in the absence of ligand (A); in the presence of 10 μM DHA (B); and in the presence of 10 μM DHA and 10 μM TCA (C and D). The sample flow rate was 5 μL/min, in (A), (B) and (C) the cone-voltage was 50 V, while in (D) it was 195 V. In all spectra the collision voltage was 4.2 V with a source block temperature of 80 °C.

Figure 6

Analysis of a RXRα with a mixture of DHA and TCA. ES mass spectra of a 10 μM RXRα solution, 10 mM ammonium acetate, pH 8.1, in the presence of an equimolar mixture of DHA and TCA (25 μM / compound) using a cone-voltage of 50 V (A); and 195 V (B), and at a collision cell voltage of 4.2 V. The sample was cleaned-up by on-line dialysis by pumping the sample solution (5 μL/min) through a dialysis fibre of molecular weight cut-off 5000, just prior to on-line ES analysis. The RXR – DHA complex is labelled with an arrow, RXRα – TCA complexes by a filled diamond, and the +178 Da modification with a filled circle. The multiple binding of TCA molecules to the protein becomes visible at the higher cone-voltage.

Figure 7

Protein binding of DHA and bile acids under non-denaturing ES conditions. ES mass spectra of a 10 μM solution of RXRα in 10 mM ammonium acetate, pH 8.1, in the absence of ligand (A); in the presence of 50 μM DHA (B); TCA (C); CDCA (D); CA (E), and LCA (F). The cone-voltage was 50 V, and the collision cell voltage 4.2 V. The sample was cleaned-up by on-line dialysis by pumping the sample solution (5 μL/min) through a dialysis fibre of molecular weight cut-off 5000, just prior to on-line ES analysis. The +178 Da modification is labelled with a filled circle, the RXRα – DHA complex by an arrow, and the RXRα - bile acid complexes with filled diamonds. Each panel shows the 13+ and 12+ charge states of monomeric RXRα LBD.

Figure 8

Bile acid – protein complexes are stable under energetic ES conditions, (cone voltage 195 V and collision cell voltage 50 V). ES mass spectra of a 10 μM solution of RXRα in 10 mM ammonium acetate, pH 8.1, in the presence of 50 μM TCA (A); 50 μM LCA (B); 50 μM LCA-3-sulphate (C); and 50 μM SDS (D). The sample was cleaned-up by on-line dialysis by pumping the sample solution (5 μL/min) through a dialysis fibre of molecular weight cut-off 5000, just prior to on-line ES analysis. The +178 Da modification is labelled with a filled circle, and the RXR – bile acid or SDS complexes with a filled diamond.

Figure 9

The high affinity agonist ligand LG268 stabilises homodimerisation of the RXRα LBD. Nano-ES mass spectra were recorded on protein samples desalted by gel filtration and diluted to ∼20 μM with 10 mM ammonium acetate, pH 8.0. Shown in (A) is the high m/z range spectrum (2000 – 6500) of apo-RXRα. Shown in (B) is the effect of the addition of two-fold excess LG268 (40 μM final concentration) to protein. Panel (C) shows the effect of adding TCA to 40 μM. Monomer (M) and dimer (D) charge states are indicated. Shown in (D-G) is the homodimer 16+ peak. The spectrum in (D) was recorded in the absence of ligand, while those in (E and F) were recorded in the presence of 40 μM LG268. The mass shift upon LG268 addition, corresponding to the mass of two molecules of LG268, is indicated by the black bar. Shown in (G) is the result of the addition of TCA to 40 μM. The spectra shown in (A-E and G) were recorded at a cone voltage of 20 V and that in (F) was at a cone voltage of 80V.