• Ligand binding site identification
• Binding site and pharmacology correlations (e.g. agonist vs. antagonist).
  
• Enabling “fragment assembly” strategies in the absence of ligand-bound X-ray structures.

Amide hydrogen/deuterium exchange, coupled with proteolysis and liquid chromatography-mass spectrometry (H/D-Ex), is gaining widespread use for the analysis of protein structure dynamics^1-3, protein–protein interactions^4,5, and protein–ligand interactions^1,6-8.

In this example the ligand binding domain of a nuclear receptor (NR), peroxisome proliferator-activated receptor gamma (PPAR-gamma), was exposed to various ligands and analyzed by H/D-Ex. NRs are a superfamily of transcriptional regulators that modulate biological processes as diverse as cellular differentiation and metabolism. Their flexibility in regard to gene regulation has been attributed to their conformational flexibility. Many studies suggest that different types of ligands induce changes in conformation and dynamics to the ligand binding domain (LBD) of PPAR-gamma. These changes consequently alter the expression profile of certain genes relating to lipid and glucose homeostasis. In fact, PPAR-gamma’s role in lipid and glucose homeostasis has made it a common target of strategies seeking to restore insulin sensitivity in patients with type II diabetes.

Here H/D-Ex was used to detect differences (perturbations) in the protein dynamics of apo and drug-bound PPAR-gamma LBD. Three different classes of drugs were surveyed: agonists, partial agonists and antagonists. The value of this technology is that H/D-Ex can be used to bin newly identified ligands (or drug-leads) into groups that elicit similar conformation or dynamic responses upon complex formation.


The rate of amide hydrogen exchange is dependent upon local fluctuations in protein structure⁹. For this reason the rate of H/D-Ex is a good indicator of regional flexibility within a protein. As illustrated above in panels A-E, ligand binding in all cases reduced the exchange rate hence stabilized structural regions closely corresponding to the crystallographically determined rosiglitazone binding site (panel E). Since ligand binding typically involves the formation of an intermolecular network involving hydrogen bonds and/or electrostatic interactions with the protein, complex formation is accompanied by stabilization. Protein stabilization is readily detectable by H/D-Ex.

In general, full agonists GW1929 and rosiglitazone (panels C-D) stabilized PPAR-gamma LBD to a greater extent than antagonist GW9662, and partial agonist nTZDpa (panels A-B). The exchange of amide hydrogens in Helix 11 and 12 (circled region) was largely impeded upon binding agonist. Both of these helices are highly dynamic in apo PPAR-gamma LBD. Upon binding antagonist or partial agonist, helices 11 and 12 appear to be as dynamic as in the unbound form. The stabilization of helix 12 in the presence of agonist supports earlier studies showing that the maintenance of the helical conformation of this helix is critical for gene activation¹⁰. These published results demonstrate the ability to predict the activities of various drugs by H/D-Ex technology ¹¹.

References:
1. Engen JR, Smith DL. Investigating protein structure and dynamics by hydrogen exchange MS. Anal Chem 2001;73:256A-265A.

2. Hamuro Y, Coales SJ, Southern MR, Nemeth-Cawley JF, Stranz DD, Griffin PR. Rapid analysis of protein structure and dynamics by hydrogen/deuterium exchange mass spectrometry. J Biomol Tech 2003;14:171-182.

3. Hoofnagle AN, Resing KA, Goldsmith EJ, Ahn NG. Changes in protein conformational mobility upon activation of extracellular regulated protein kinase-2 as detected by hydrogen exchange. Proc Natl Acad Sci U S A 2001;98:956-961.

4. Baerga-Ortiz A, Hughes CA, Mandell JG, Komives EA. Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein. Protein Sci 2002;11:1300-1308.

5. Ehring H. Hydrogen exchange/electrospray ionization mass spectrometry studies of structural features of proteins and protein/protein interactions. Anal Biochem 1999;267:252-259.

6. Englander JJ, Del Mar C, Li W, Englander SW, Kim JS, Stranz DD, Hamuro Y, Woods VL, Jr. Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry. Proc Natl Acad Sci U S A 2003;100:7057-7062.

7. Hamuro Y, Wong L, Shaffer J, Kim JS, Stranz DD, Jennings PA, Woods VL, Jr., Adams JA. Phosphorylation driven motions in the COOH-terminal Src kinase, CSK, revealed through enhanced hydrogen-deuterium exchange and mass spectrometry (DXMS). J Mol Biol 2002;323:871-881.

8. Yan X, Broderick D, Leid ME, Schimerlik MI, Deinzer ML. Dynamics and ligand-induced solvent accessibility changes in human retinoid X receptor homodimer determined by hydrogen deuterium exchange and mass spectrometry. Biochemistry 2004;43:909-917.

9. Englander SW, Kallenbach NR. Hydrogen exchange and structural dynamics of proteins and nucleic acids. Q Rev Biophys 1983;16:521-655.

10. Johnson BA, Wilson EM, Li Y, Moller DE, Smith RG, Zhou G. Ligand-induced stabilization of PPARgamma monitored by NMR spectroscopy: implications for nuclear receptor activation. J Mol Biol 2000;298:187-194.

11. Hamuro Y, Coales SJ, Morrow JA, Molnar KS, Tuske SJ, Southern MR, Griffin PR. Hydrogen/deuterium-exchange (H/D-Ex) of PPARgamma LBD in the presence of various modulators. Protein Sci 2006;15:1883-1892.

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