THE DYNAMICS AND MISFOLDING PATHWAYS OF THE PRION PROTEIN AT ATOMIC RESOLUTION Chin Jung Cheng A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2015 Reading Committee: Valerie Daggett, Chair James Bryers Shaoyi Jiang Program Authorized to Offer Degree: Bioengineering © Copyright 2015 Chin Jung Cheng University of Washington Abstract THE DYNAMICS AND MISFOLDING PATHWAYS OF THE PRION PROTEIN AT ATOMIC RESOLUTION Chin Jung Cheng Chair of the Supervisory Committee: Professor Valerie Daggett Bioengineering Prion diseases are fatal, transmissible and incurable neurodegenerative diseases that only infect mammals. While the manifestation of prion disease is not completely understood, it is known that the prion protein (PrP) plays a critical role in prion disease development. The PrP can exist in two different isoforms: PrP C is the native cellular form which is innocuous, while PrP Sc is the pathogenic form. PrP Sc is aggregation-prone thus forming soluble oligomers which are toxic and infectious. Using molecular dynamics simulations, we have investigated misfolding pathways for human PrP pathogenic mutants and bovine PrP at acidic pH. In order to better understand the molecular dynamics of the PrP C at a physiological environment, we have also performed simulations of the human PrP C attached with glycans in a membrane environment, revealing protective mechanisms against misfolding. Using recent experimental data on PrP Sc soluble oligomers, we have validated our current structural model for PrP Sc oligomers and finally, combining our findings from both bovine PrP Sc and human PrP C, we have constructed a system to model the PrP Sc -induced misfolding for the human PrP C in a physiological environment. Our research sheds light on the PrP misfolding mechanism at the atomic level, potentially advancing future therapeutic and diagnostic development on prion diseases. TABLE OF CONTENTS Chapter 1 ....................................................................................................................................... 8 Background and significance ....................................................................................................... 8 1.1 Prion Diseases ................................................................................................................. 8 1.2 The Prion Protein ............................................................................................................ 9 1.3 Methods of Studying Prion Diseases ............................................................................ 11 1.4 Spiral Model for Oligomeric PrP Sc ............................................................................... 14 1.5 This Work ..................................................................................................................... 15 Chapter 2 ..................................................................................................................................... 20 Human Prion Protein Pathogenic Mutations: Y218N & E196K ............................................ 20 2.1 Summary ....................................................................................................................... 20 2.2 Introduction ................................................................................................................... 21 2.3 Results ........................................................................................................................... 23 2.4 Discussion ..................................................................................................................... 28 2.5 Materials and Methods .................................................................................................. 33 Chapter 3 ..................................................................................................................................... 44 Misfolding of bovine prion protein at acidic pH ...................................................................... 44 3.1 Summary ....................................................................................................................... 44 3.2 Introduction ................................................................................................................... 44 3.3 Results and Discussion ................................................................................................. 47 3.4 Materials and Methods .................................................................................................. 58 3.5 Conclusions ................................................................................................................... 61 Chapter 4 ..................................................................................................................................... 70 Simulations of Membrane-bound Diglycosylated Human Prion Protein .............................. 70 4.1 Summary ....................................................................................................................... 70 4.2 Introduction ................................................................................................................... 70 4.3 Results and Discussion ................................................................................................. 73 4.4 Conclusions ................................................................................................................... 82 4.5 Materials and Methods .................................................................................................. 82 Chapter 5 ................................................................................................................................... 102 Comparison of structural models of the prion oligomer ....................................................... 102 5.1 Summary ..................................................................................................................... 102 5.2 Introduction ................................................................................................................. 103 5.3 Materials and Methods ................................................................................................ 105 5.4 Results and Discussion ............................................................................................... 107 5.5 Conclusions ................................................................................................................. 117 Chapter 6 ................................................................................................................................... 136 1 Model system for prion propagation ....................................................................................... 136 6.1 Summary ..................................................................................................................... 136 6.2 Material and Methods ................................................................................................. 137 6.3 Results and Discussion ............................................................................................... 140 2 LIST OF FIGURES Figure 1.1 PrP C membrane-bound via GPI anchor. ...................................................................... 16 Figure 1.2 Illustration of the seeded-nucleation mechanism. ....................................................... 17 Figure 1.3 Example of an MD-derived monomeric PrP Sc . ........................................................... 18 Figure 1.4 The spiral model. ......................................................................................................... 19 Figure 2.1 Native structure of the human PrP............................................................................... 35 Figure 2.2 Cα RMSD of the globular domain (residues 128-228) and HA (residues 144-156) for Y218N and E196K simulations (three simulations of each)................................................. 36 Figure 2.3 HA detachment from the PrP core in Y218N simulation 3 ......................................... 37 Figure 2.4 HA detachment from the PrP core in E196K simulation 1 ......................................... 38 Figure 2.5 Solvent accessible surface area of the hydrophobic core and F198 in the Y218N and E196K simulations ................................................................................................................ 39 Figure 2.6 Salt bridge network around the F198 side chain ......................................................... 40 Figure 2.7 Change in interhelical angle between HB and HC in Y218N ..................................... 41 Figure 2.8 Loss of hydrophobic packing around in the X-loop in Y218N simulations ................ 42 Figure 2.9 Formation of nonnative strand (E1) in both Y218N and E196K simulations ............. 43 Figure 3.1 Native structure of bovine PrP C (residues 90–231) ..................................................... 63 Figure 3.2 Cα root-mean-square deviation (RMSD) and root mean square fluctuations (RMSF) of the bovine PrP simulations ............................................................................................... 64 Figure 3.3 Conformational changes of bovine PrP at mid and low pH ........................................ 65 Figure 3.4 Changes in polar contacts at different pH ................................................................... 66 Figure 3.5 Disruption of the hydrophobic core ............................................................................