• Ligand binding site identification
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• 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^1,2, protein–protein interactions^3,4, and protein–ligand interactions^5-10.

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^5-10. 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 structure⁵. 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 activation. These published results demonstrate the ability to predict the activities of various drugs by H/D-Ex technology.

References:
1. 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.

2. Yoshitomo Hamuro, Kathleen S. Molnar, Stephen J. Coales, Bo Ou Yang, Alana Simorellis, and Thomas C. Pochapsky "Hydrogen-Deuterium Exchange Spectrometry for Investigation of Backbone Dynamics of Oxidized and Reduced Cytochrome P450_cam" J. Inorg. Biochem. 2008, 102, 364.

3. James R. Horn, Brian Kraybill, Elizabeth J. Petro, Stephen J. Coales, Jeffrey A. Morrow, Yoshitomo Hamuro and Anthony A. Kossiakoff "The role of protein dynamics in increasing the binding affinity of an engineered protein-protein interaction established by H/D exchange mass spectrometry" Biochemistry 2006, 45, 8488-8498.

4. Stephen J. Coales, Steven J.Tuske, Justine C. Tomasso, and Yoshitomo Hamuro “Epitope Mapping by Amide Hydrogen/Deuterium Exchange Coupled with Proteolysis and Liquid Chromatography Mass Spectrometry” Rapid Communications in Mass Spectrometry 2009 Mar;23(5):639-47. 

5. 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.

6. Yoshitomo Hamuro, Stephen J. Coales, Jeffrey A. Morrow, Kathleen S. Molnar, Steven J, Tuske, Mark R. Southern, and Patrick R. Griffin "Hydrogen/Deuterium-Exchange (H/D-Ex) of PPARγ LBD in the Presence of Various Modulators" Protein Sci. 2006, 15, 1-10.

7. Lusong Luo, Jeffrey D. Carson, Kathleen S. Molnar, Steven J. Tuske, Stephen J. Coales, Yoshitomo Hamuro, Chiu-mei Sung, Valery Sudakin, Kurt R. Auger, Dashyant Dhanak, Jeffrey R. Jackson, Pearl S. Huang, Peter J. Tummino, Robert A Copeland "Conformation-Dependent Ligand Regulation of ATP Hydrolysis by human KSP: Activation of Basal Hydrolysis and Inhibition of Microtubule-Stimulated Hydrolysis by a Single, Small Molecule Modulator" J. Am. Chem. Soc 2008, 130, 7584-7591. 

8. Gregory J. Kornhaber, Michael B. Tropak, Gustavo Maegawa, Stephen J. Tuske, Stephen J. Coales, Don J. Mahuran, Yoshitomo Hamuro "Isofagomine Induced Stabilization of Glucocerebrosidase" ChemBioChem 2008 Nov 3;9(16):2643-9

9. Michael B. Tropak, Gregory Kornhaber, Brigitte Rigat, Gustavo Maegawa, Justin Buttner, Jan Blanchard, Cecilia Murphy, Steven J. Tuske, Stephen J. Coales, Yoshitomo Hamuro, Eric Brown, Don Mahuran "Identification of Pharmacological Chaperones for Gaucher Disease and Characterization of their Effects on b-Glucocerebrosidase by Hydrogen/Deuterium Exchange Mass Spectrometry" ChemBioChem 2008 Nov 3;9(16):2650-62

10. Vikas Chandra, Pengxiang Huang, Yoshitomo Hamuro, Srilatha Raghuram, Yongjun Wang, Thomas P. Burris, Fraydoon Rastinejad "Structural Organization of the Intact PPARγ-RXRα Nuclear Receptor Complex on DNA"  Nature 2008 Nov 20;456(7220):350-6 

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