1.  The role of H293 in Protein Arginine Methyltransferases 1 (PRMT1) Brittany Boykin Auburn University Department of Chemistry and Biochemistry Computational Seminar National Organization for the Professional Organization of Black Chemist and Chemical Engineers 2015 Annual Conference Orlando, Florida 24, September 2015 1

2. Outline Background Overall Goal Future Work Acknowledgments Q/A Current Study 2

3. Post-Translational Modification (PTMs) Remarkably, there are 200+ types of PTM’s that include kinases, phosphatases, transferases Walsh C. (2006) Posttranslational Modification of Proteins: Expanding Natures Inventory. Englewood, Colo.: Roberts and Co. Publishers. Xxi, 490 p. p. 3

4. Arginine Methylation FASEB J. 10, 471-480 (1996) Methylation reactions involve methyl group transfers (AdoMet is the methyl donor and this reaction displays a Sn2 type geometry) 4

5. Protein Arginine Methyltransferases (PRMTs) Bedford, M. T., and Clarke, S. G. (2009) Protein Arginine Methylation in mammals: Who, What, and why. Mol Cell 33, 1-13 5

6. Physiological roles of PRMTs Transcriptional Activity RNA splicing and transport DNA repair Chromatin Remodeling Signal Transduction Neuronal Cell Differentiation Virus-host Biochemistry Journal of Biological Chemistry Vol. 289, NO. 13, pp. 9320-9327, March 28, (2014) PRMTs regulate proteins in cell processes and human diseases 6

7. Target Diseases 7

8. PRMT Human Isoforms Cell. Mol. Life Sci. 2009 66:2109. PRMT1 8

9. Human Diseases: PRMT1’s Target 9

10. Structure of PRMT1 Structure 2003 11: 509. Dimerization - essential for SAM binding and enzymatic activity 10

11. Active Site of PRMT1 Biochemistry 2011, 50, 3332-3345J. Bio. Chem. Vol. 289, NO. 13, pp. 9320-9327 2014 11

12. Overall Goal Product Specificity Org. Biomol. Chem., 2015, 13, 549-560 Control Sterics & Nucleophilicity What use of the product depends on the local conditions? We want to gain more insight by dissecting the active site of PRMT1 and identifying the significance of specific residues in regards to the substrate What orientation does the substrate display with the H293S mutation? 12

13. Importance of H293 Salt-Bridge Biochemistry 2011 April 26; 50(16): 3332-3345 This short bond plays a critical role in forming the two-helix boundary that impact cofactor and peptide binding 13

14. Alternative Mechanism 14

15. Current Research Objective Objective Product Specificity of H293: Mutant H293S; How does the protein environment influence product specificity using aMD simulations and QM/MM calculations Analyzed systems: H293S-Arg H293S-MMA-ADMA H293S-MMA-SDMA With the Ser in place of His: What orientation does the substrate display Distribution of the His vs Ser Question: Does H293 have more affect on the active site than proposed? 15

16. Assisted Model Building With Energy Refinement (AMBER) 16

17. Molecular Dynamics (MD) F = ma 17

18. (QM) | (MM) Quantum Mechanical (QM)  Electronic Processes  Bonding Breaking/formation  DFT/ Ab Initio  Primary subsystem (PS) Molecular Mechanical (MM)  Force-field based method  Computationally Efficient  Secondary subsystem (SS) Combined QM/MM  Chemical Reaction in macromolecules 18

19. PRMT1 RMSF Analysis 19

20. Preferred Methylation SDM A ADMA SDM A H293S-MMANη2 H293S-MMANη1 20

21. Future Work To continue the QM/MM simulations for all mutant complexes: H293S-Arg, H293S-MMA-proADMA, and H293S- MMA-proSDMA Continue to compare to double mutant (H293S-M48F) and WT-enzyme To compare to experimental results 21

22. Acknowledgements o Alabama Super Computer Center o Huntsville Alabama o Collaborators: Dr. Joan Hevel (Utah State University) o Orlando Acevedo o My Lab members o Symon Gathiaka o Nicole Ippolito o Robel Ghebreab o Brian Doherty 22

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