A two-stage differential hydrogen deuterium exchange method for the rapid characterization of protein/ligand interactions

Abstract

The peroxisome proliferator-activated receptor is a member of the nuclear receptor superfamily of transcriptional regulators. Regulation of the nuclear receptors occurs through changes to the structure and dynamics of the ligand-binding domain. Therefore, the need has arisen for a rapid method capable of detecting changes in the dynamics of nuclear receptors following ligand binding. We recently described how solution-phase amide hydrogen/deuterium exchange (HDX) provides a biophysical technique for probing changes in protein dynamics induced by ligand interaction. Building from this platform, we have optimized the robustness of the differential HDX experiment by minimizing systematic errors, and have increased the efficiency of the chromatographic separation through the use of high-pressure liquid chromatography. Using knowledge gained previously from comprehensive HDX experiments of PPARgamma, a modest throughput method to probe changes in the dynamics of key regions of the receptor was developed. A collection of ten synthetic and endogenous PPARgamma ligands were characterized with this new method requiring approximately 24 h of analysis. This is a dramatic improvement over the 10 d of analysis that would have been required with our previous approach for comprehensive differential HDX analysis. In addition to demonstrating the utility of this approach, the study presented here is the first to measure changes to the dynamics of PPARgamma upon the binding of putative endogenous ligands.

FIGURE 1

Evidence for systematic error in linear HDX experiments. Percent deuterium incorporation plotted against log time (s) for six different regions of the PPARγLBD. Data were acquired at day 1 (solid black line), day 5 (solid grey line) and month 2 (broken black line). The number in parentheses represents the charge state of the ion. Systematic errors are most obvious after the 2-mo interval; however, they can be observed between day 1 and day 5 for regions 391–401 and 443–452. Differences between the day 1 and day 5 data were also observed—for example, at the 900-s and 3600-s time points of 391–401 (+2).

FIGURE 2

Differential HDX analysis of PPARγ LBD ± nTZDpa with randomized order of time-point acquisition. Percent deuterium incorporation plotted versus log time (s) for six different regions of the receptor. Each plot shows the HDX data for ligand-bound and free receptor. These data were generated with a randomized experimental sequence in which the on-exchange of apo- and ligand-bound receptor were acquired in parallel. Each data point represents the mean of four individual on-exchange experiments (not re-measurement of a single on-exchange experiment) and the error bars represent the standard deviation in the measurement. A two-way ANOVA was performed for each region of the receptor (*** = P value < 0.001, NS = not significant, P value > 0.05).

FIGURE 3

Differential HDX of PPARγ ± six synthetic ligands with randomized order of time-point acquisition. Six log versus time plots are shown for the region of the receptor spanning residues 299–309. Significant changes in HDX rate were observed for nTZDpa, MRL-20, MRL-24, and GW1929. No change was observed for rosiglitazone or BVT.13 (*** = P value < 0.001, NS = not significant, P value > 0.05).

FIGURE 4

HPLC ESI MS analysis of the pepsin digest of PPARγ LBD. Total ion chromatogram showing the separation of the proteolytic peptides obtained from the pepsin digestion of the PPARγ-LBD. The gradient was from 2% CH₃CN to 40% CH₃CN over 10 min with a flow rate of 250 μL/min. Extracted ion chromatograms are shown for the [443–452 +2H]²⁺, [391–401 +2H]²⁺, and [453–477 +3H]³⁺ ions. Although baseline resolution of all the peptides in the mixture was not possible with the 50 mm x 2.1 mm column containing 1.9- μm-diameter particles, the resolution (½ height ~ 6 sec) was sufficient to ensure each of the 31 peptide ions of interest was not overlapped with any other ions during the mass analysis. To obtain the equivalent resolution of these peptides with a column packed with 5-μm particles required a 15-min gradient.

FIGURE 5

Single on-exchange time-point differential HDX data. Data from the single on-exchange time-point differential HDX analysis of PPARγLBD ± rosiglitazone, MRL-20, and BVT-13. The top row shows the reduction in HDX rate for residues 279–287 located in H3. Residues 470–477 (bottom row) are located in H12 and show stabilization with rosiglitazone and MRL-20 but not with BVT.13.

FIGURE 6

Single on-exchange time-point differential HDX data for the endogenous ligands 15PGJ2, 15(S)-HETE, 9(S)-HODE, and 13(S)-HODE overlaid onto the structure of PPARγLBD (2PRG.pdb).