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Fxr Agonists Induce Distinct H-12 Structural States University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 2021 FXR AGONISTS INDUCE DISTINCT H-12 STRUCTURAL STATES Vikash Kumar Follow this and additional works at: https://scholarworks.umt.edu/etd Part of the Biochemistry Commons, Biophysics Commons, and the Molecular Biology Commons Let us know how access to this document benefits ou.y Recommended Citation Kumar, Vikash, "FXR AGONISTS INDUCE DISTINCT H-12 STRUCTURAL STATES" (2021). Graduate Student Theses, Dissertations, & Professional Papers. 11760. https://scholarworks.umt.edu/etd/11760 This Thesis is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. FXR AGONISTS INDUCE DISTINCT H-12 STRUCTURAL STATES By VIKASH KUMAR Thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry and Biophysics The University of Montana Missoula, MT June 2021 Approved by: Scott Whittenburg, Dean of The Graduate School Graduate School Bruce Bowler, Chair Chemistry and Biochemistry Travis Hughes, Advisor Biomedical and Pharmaceutical Sciences Steve Sprang Division of Biological Sciences J.B. Alexander (Sandy) Ross Chemistry and Biochemistry Jesse Hay Division of Biological Sciences i Kumar, Vikash, MS, Spring 2021 Biochemistry and Biophysics FXR AGONISTS INDUCE DISTINCT H-12 STRUCTURAL STATES Chairperson: Bruce Bowler Thesis Advisor: Travis Hughes The nuclear receptor ligand-binding domain (LBD) is a highly dynamic entity. The FXR LBD shows multiple low-energy conformational states of the activation function-2 (AF-2) coregulator binding surface upon ligand binding, indicating the complexity of FXR activation. However, it is unknown how ligand binding leads to different conformational states within the AF-2 region centered on helix 12 (H-12) of the LBD. Here we observe the conformation of the coregulator binding surface (H-12 specifically) of FXR upon ligand binding in solution using fluorine-19 (19F) nuclear magnetic resonance (NMR) and simulations of this surface using molecular dynamics. Fluorescence anisotropy of fluorescein-labeled coregulator peptides reveals a correlation between structural conformations of the coregulator binding surface and the function of FXR. While the coregulator surface of apo FXR and partial-agonist bound FXR exchanges between multiple low energy conformations, full-agonist bound FXR is restricted to few conformations, which favor coactivator binding. Furthermore, we find that two ligands that induce similar affinities for a coactivator peptide have distinct coregulator binding surface structures. ii TABLE OF CONTENTS Page No. LIST OF TABLES -----------------------------------------------------------------------------------v LIST OF FIGURES ---------------------------------------------------------------------------------vi CHAPTERS I. INTRODUCTION---------------------------------------------------------------------1 1. Nuclear Receptor (NR)---------------------------------------------------------------------3 1.1 NR Structure-----------------------------------------------------------------------------3 1.2 NRs structure-function relation--------------------------------------------------------6 1.3 NR classification------------------------------------------------------------------------6 1.4 NR as drug target------------------------------------------------------------------------8 2. Farnesoid X Receptor (FXR) --------------------------------------------------------------8 2.1 FXR expression--------------------------------------------------------------------------9 2.2 FXR as drug target----------------------------------------------------------------------9 2.3 FXR structure--------------------------------------------------------------------------10 2.4 FXR: DNA binding--------------------------------------------------------------------12 2.5 FXR activation: ligand and coactivator binding-----------------------------------13 2.6 FXR ligands----------------------------------------------------------------------------15 2.7 Coactivator peptides: SRC -----------------------------------------------------------18 3. Results --------------------------------------------------------------------------------------19 3.1 FXR purification and activity--------------------------------------------------------19 iii 3.2 The efficacy of different FXR ligands in coregulator peptide recruitment----21 3.3 H-12 conformational change observed by 19F-NMR-----------------------------24 3.4 Simulation confirms the 19F-NMR results-----------------------------------------33 3.5 Discussion------------------------------------------------------------------------------40 3.6 Conclusion------------------------------------------------------------------------------41 3.7 Future direction------------------------------------------------------------------------42 4. Methods-------------------------------------------------------------------------------------45 4.1 Protein purification--------------------------------------------------------------------45 4.2 FXR-LBD sequence used in this work----------------------------------------------46 4.3 Site-directed mutagenesis-------------------------------------------------------------46 4.4 Fluorescence Anisotropy (FA) assay------------------------------------------------47 4.5 Preparation of 19F-NMR samples---------------------------------------------------47 4.6 Molecular dynamics simulation------------------------------------------------------49 REFERENCES -------------------------------------------------------------------------------------50 iv LIST OF TABLES Table Page No. 1. SRC peptides and their sequences--------------------------------------------------------19 2. Coactivator peptide affinity for FXR in the presence of ligands---------------------23 3. List of the significant peak, size, and percentage of each spectrum in Fig. 16----- 30 4. cMD builds and its corresponding PDB files-------------------------------------------34 5. Primer sequences used for mutagenesis-------------------------------------------------46 v LIST OF FIGURES Figure Page No. 1. Domain structure of NRs-------------------------------------------------------------------3 2. NR DNA binding domain------------------------------------------------------------------4 3. Cartoon representation of structurally conserved NR LBD---------------------------5 4. NRs signaling mechanism------------------------------------------------------------------7 5. Schematic diagram of FXR---------------------------------------------------------------11 6. Model structure of FXR DBD and LBD------------------------------------------------12 7. FXR binds to DNA either as a heterodimer with RXR or as a monomer to regulate the expression of various genes----------------------------------------------------------13 8. Mousetrap model of FXR-LBD activation---------------------------------------------15 9. Chemical structure of FXR ligands used-----------------------------------------------17 10. Effect of MBP tag cleavage on FXR-LBD activity-----------------------------------20 11. SDS page gel of size exclusion purified MBP-FXR----------------------------------21 12. Efficacies of FXR ligands----------------------------------------------------------------22 13. Efficacies of antagonists of FXR--------------------------------------------------------24 14. The location of cysteines on the FXR-LBD--------------------------------------------26 15. Mutations in FXR-LBD bound to ivermectin to verify the source of peaks-------27 16. 19F-NMR result of C432H and Double mutant---------------------------------------28 17. Deconvoluted C432H-DM delta spectra------------------------------------------------29 18. C432H-DM difference spectrum of MBP-FXR-LBD-BTFA ----------------------31 19. Addition of peptide, SRC 1-2 consolidates the two H-12 signal -------------------32 vi 20. Cleaving the MBP tag does not affect the 19F-NMR signal-------------------------33 21. MD simulations of wt FXR-LBD apo and bound to different ligands--------------35 22. Histogram of H-12 RMSD---------------------------------------------------------------38 23. RMSD fluctuations of ligand bound to LBP of FXR-LBD--------------------------39 24. H-3 RMSD of FXR when bound to different ligands---------------------------------43 25. Position of residues on H-3 for single mutations--------------------------------------44 vii CHAPTER I INTRODUCTION Farnesoid X receptor (FXR) is a ligand-activated transcription factor,1 found mainly in the liver and kidney2. FXR maintains the homeostasis of bile acids, vital for metabolic regulation and liver protection by regulating bile acids3. Like other nuclear receptors (NR) superfamily members, FXR recruits coregulators upon ligand binding, thereby changing the expression level of genes involved in bile acid, glucose, and lipid metabolism. Because it regulates bile acids in the liver, FXR is a potential drug target for treating several liver- related disorders, including nonalcoholic steatohepatitis (NASH) and primary biliary cholangitis (PBC), and diabetes4,5. Partial FXR activation is a promising strategy to target liver disorders and to reduce mechanism-based side effects. Partial Nuclear
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