Predicting the Pose of β-Casomorphin-5 and 7 in the Opioid Receptors Christopher Oberc, Bachelor of Science Submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Faculty of Mathematics & Sciences, Brock University St. Catharines, Ontario ©2015 Abstract The opioid receptors consist of three main subtypes; μ, δ, and κ. Previous binding studies have shown that fragments of the milk protein, β-casein, known as β-casomorphins are agonists of these receptors which are selective for the μ receptor subtype. Using the crystal structures of these three receptors, computational molecular docking studies were done using the software GOLD to determine the conformation of β-casomorphin-5 and 7 when they bind to these three opioid receptors. GOLD was able to discriminate among the three receptors when docking the rigid ligands co-crystalized with the receptors. However, GOLD could not discriminate among the three receptors for either of the highly flexible β-casomorphins. A per amino acid scoring method was developed to overcome this problem. This method was used to predict the conformation of both β-casomorphin-5 and 7 in the μ receptor and determine that the two amino acid residues, Lys303 and Trp318 of the μ receptor are responsible for discriminating among the three receptor subtypes for binding of the β-casomorphin-5 and 7. i Acknowledgements I would like to thank Heather Gordon and Hongbin Yan for supporting me throughout my graduate degree. I would also like to thank Jeffery Atkinson and Randall Dumont for their contributions. I am also grateful for Michelle Eisner whom has assisted me throughout my degree. Finally, I would like to thank any other faculty members and students that have contributed towards my degree. ii Table of Contents Abstract ................................................................................................................................ i Acknowledgements ............................................................................................................. ii Table of Contents ............................................................................................................... iii List of Figures ............................................................................................................... vii List of Tables ................................................................................................................. xv List of Abbreviations ................................................................................................... xvii Introduction ......................................................................................................................... 1 1: Opioid Receptors ......................................................................................................... 1 1a: Function and Biological Relevance ....................................................................... 1 1b: Physiological Function of Opioid Receptors ......................................................... 8 1c: Amino Acid Sequence of the WT Receptors ....................................................... 11 1d: X-Ray Crystal Structures of Proteins .................................................................. 14 1e: Protein Data Bank and Limitations of X-Ray Structures .................................... 15 1f: Opioid Receptor-Lysozyme Chimera .................................................................. 16 1g: Opioid Receptor Ligands ..................................................................................... 24 2: Opioid Peptides ......................................................................................................... 28 2a: Endogenous Opioid Receptor Peptide Ligands ................................................... 28 iii 2b: Exogenous Opioid Receptor Peptide Ligands ..................................................... 28 3: Computational Molecular Docking ........................................................................... 32 3a: Genetic Algorithm ............................................................................................... 32 3b: Genetic Optimization for Ligand Docking (GOLD) ........................................... 37 3c: Scoring Function .................................................................................................. 39 3d: Root Mean Squared Deviation ............................................................................ 48 3e: Rescoring ............................................................................................................. 49 Materials and Methods ...................................................................................................... 50 1: Crystal Structure Modifications ................................................................................ 51 2: Binding Site Parameters ............................................................................................ 53 3: GA Parameters .......................................................................................................... 54 4: Flexible Side chains .................................................................................................. 54 Results ............................................................................................................................... 56 1: Receptor Superpositioning ........................................................................................ 56 2: Native Docking ......................................................................................................... 58 2a: δ Receptor ............................................................................................................ 58 2b: µ Receptor ........................................................................................................... 63 2c: κ Receptor ............................................................................................................ 66 2d: 50Å Radius Docking ........................................................................................... 68 3: Non-Native Cross Docking ....................................................................................... 72 iv 3a: Naltrindole ........................................................................................................... 72 3b: β-FNA .................................................................................................................. 73 3c: JDTic.................................................................................................................... 75 4: β-Casomorphin Docking ........................................................................................... 76 4a: β-Casomorphin-7 ................................................................................................. 76 4b: β-Casomorphin-5 ................................................................................................. 80 5: DAMGO Docking ..................................................................................................... 85 5a: Traditional Scoring Analysis ............................................................................... 85 5b: Per Amino Acid Scoring Analysis ...................................................................... 87 5c: Conclusion Concerning DAMGO Docking ......................................................... 94 6: Per Amino Acid Scoring Analysis of β-Casomorphin-5........................................... 94 6a: Rescoring of Poses of β-casomorphin-5 from the µ Receptor Docking Experiments in δ and κ Receptors ......................................................................................... 95 6b: Mutant µ Receptor Rescoring .............................................................................. 99 7: Per Amino Acid Scoring Analysis of β-Casomorphin-7 Docked to μ Receptor .... 102 7a: Rescoring μ Poses to δ and κ Receptors ............................................................ 103 7b: µ Mutant Rescoring Analysis ............................................................................ 115 Discussion ....................................................................................................................... 135 1: Non-Native β-FNA Docking ................................................................................... 135 2: DAMGO Pose Analysis .......................................................................................... 142 v 3: β-Casomorphin-5 Pose 268 in the W318Y Mutant ................................................. 143 4: β-Casomorphin-7 Pose Selection ............................................................................ 144 5: Global Receptor Conformation ............................................................................... 147 Conclusion ...................................................................................................................... 151 References ....................................................................................................................... 153 Appendix ......................................................................................................................... 159 vi List of Figures Figure 1: Chemical structure of morphine and dihydromorphine-[7,8-3H]. ................................... 2 Figure 2: Structure of MCOPPB ..................................................................................................... 4 Figure 3: Structural determination of rhodopsin.