Natalie Elizabeth Goldberger

My Story:

Using Mass Spectroscopy to Study the Surface Conformation of the Androgen Receptor When Bound to Pharmacologically Diverse Ligands

The full-length crystal structure of the androgen receptor (AR) has unfortunately remained elusive even though the AR ligand binding domain (LBD) while bound to the agonists R18811 and DHT2 has been solved. The crystal structure of full-length AR while bound to pharmacologically diverse ligands would be extremely useful because it might provide a glimpse in to each ligands mechanism of AR activation and inactivation. In order to circumvent the problems associated with trying to crystallize full-length AR, our lab is using ESI MS/MS in combination with the lysine modifying reagent NHS-biotin to obtain surface topology information about AR when bound to an AR agonist (DHT), antagonist (bicalutamide), and SARM (S-4)3. SARMs are selective androgen receptor modulators that are unique because of their ability to activate AR in a tissue selective manner. Our method of studying AR surface topology was adapted from a paper by Kvaratskhelia, et al.4 in which NHS-biotin is used to biotinylate surface accessible lysine residues: the biotinylated lysines are detected by mass spectroscopy because of the 226.3 Da mass shift produced by the biotin addition.

At this point, isolation of full-length AR bound to ligands other than DHT has been a challenge because AR is not as stable when bound to these other ligands. Thus, most of the mass spectroscopy data generated so far is from HSA (a positive control) and AR bound to DHT (AR-DHT). With HSA we have achieved 80% sequence coverage and detected 25 biotinylated lysines using mass spectroscopy, yet with AR-DHT we have achieved only 22% sequence coverage and have detected no biotinylated lysines with certainty. However, once the biotinylation, protein digestion, and protein purification methods have been optimized, we hope to observe unique biotinylation patterns for AR depending on the specific ligand bound. These biotinylation pattern differences may provide a clue about how each ligand is able to achieve pharmacologically distinct AR activity through biochemical mechanisms of ligand-induced conformational changes. Most of all, we hope this research will provide a better understanding of how SARMs are able to interact with AR in a tissue specific manner, perhaps by producing distinct SARM-induced conformational changes in AR which promote the recruitment of unique sets of cofactors.

1 Matias, P.M., et al. J Biol Chem. 2000 Aug 25; 275(34): 26164-71.

2 Sack, J.S., Proc Natl Acad Sci USA. 2001 Apr 24; 98(9): 4904-9.

3 Yin, D., et al. J Pharmacol Exp Ther. 2003 Mar; 304(3): 1334-40.

4 Kvaratskhelia, M., et al. Proc Natl Acad Sci USA. 2002 Dec 10;99(25):15988-93.