Investigation of ligand interactions with human RXRalpha by hydrogen/deuterium exchange and mass spectrometry.

Several different agonists of the retinoic X receptor alpha (hRXRalpha) were examined for their effects on the amide H/D exchange kinetics of the homodimeric protein using mass spectrometry. Some agonists, LG 100268, SR11246, and DHA, bind such that slower deuterium exchange-in occurs compared with 9-cis-retinoic acid (9-cis-RA), whereas others, fenretinide and methoprenic acid, result in poorer protection during binding and hence faster exchange-in. Protection against H/D exchange by different agonists and the inhibition of H/D exchange kinetics relative to 9-cis-RA varies markedly in different regions of the protein. Agonists LG 100268, SR11246, and DHA generally inhibit faster exchange processes in the ligand binding regions of hRXRalpha than does the native ligand 9-cis-RA. In at least half of these regions, the level of protection by 9-cis-RA lags behind the agonists even after 60 min. Methoprenic acid did not significantly protect hRXRalpha against amide hydrogen exchange. An efficient method is described for comparing the effects of different agonists on the protein structure of the agonist-RXRalpha complex.

Global analysis of three-state protein unfolding data.

A new method for analyzing three-state protein unfolding equilibria is described that overcomes the difficulties created by direct effects of denaturants on circular dichroism (CD) and fluorescence spectra of the intermediate state. The procedure begins with a singular value analysis of the data matrix to determine the number of contributing species and perturbations. This result is used to choose a fitting model and remove all spectra from the fitting equation. Because the fitting model is a product of a matrix function which is nonlinear in the thermodynamic parameters and a matrix that is linear in the parameters that specify component spectra, the problem is solved with a variable projection algorithm. Advantages of this procedure are perturbation spectra do not have to be estimated before fitting, arbitrary assumptions about magnitudes of parameters that describe the intermediate state are not required, and multiple experiments involving different spectroscopic techniques can be simultaneously analyzed. Two tests of this method were performed: First, simulated three-state data were analyzed, and the original and recovered thermodynamic parameters agreed within one standard error, whereas recovered and original component spectra agreed within 0.5%. Second, guanidine-induced unfolding titrations of the human retinoid-X-receptor ligand-binding domain were analyzed according to a three-state model. The standard unfolding free energy changes in the absence of guanidine and the guanidine concentrations at zero free-energy change for both transitions were determined from a joint analysis of fluorescence and CD spectra. Realistic spectra of the three protein states were also obtained.

Dynamics and ligand-induced solvent accessibility changes in human retinoid X receptor homodimer determined by hydrogen deuterium exchange and mass spectrometry.

Receptors for retinoic acid act as ligand activated transcription factors. The three-dimensional structure of the retinoid X receptor (RXR) ligand binding domain has been determined, but little information is available concerning the properties of the protein in solution. Hydrogen/deuterium exchange followed by electrospray ionization mass spectrometry was used to probe the solution conformation of the recombinant human RXRalpha homodimer ligand binding domain in the presence and absence of 9-cis-retinoic acid (9-cis-RA). Within the experimental time domain (0.25-180 min), about 20 amide hydrogens showed decreased exchange rates in the presence of saturating concentrations of 9-cis-RA as compared to those found for the homodimer in the absence of ligand. Most of the amides were located in peptides derived from regions of the protein shown by the X-ray structure to interact with the bound ligand: the amino termini of helices 3 and 9, the two beta sheets, helix 8, the H8-H9 loop, and the carboxyl terminus of helix 11. Unexpectedly, protection was also observed in peptides derived from helices 7, 10, 11, and the H7-H8 and H10-H11 loops, regions that are not directly in contact with bound 9-cis-RA. These results suggest that the binding of ligand results in additional effects on the conformation or dynamics of the homodimer in solution as compared to those observed for the X-ray structure. Overall, the change in deuterium exchange induced by the binding of 9-cis-RA correlated reasonably well with changes in hydrogen bonding, residue depth, and/or solvent accessibility predicted from the crystal structure.