UNIVERSITY OF CALIFORNIA Los Angeles Protein structure in reversible amyloid formed by low-complexity regions A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biological Chemistry by Michael Patrick Hughes 2018 © Copyright by Michael Patrick Hughes 2018 ABSTRACT OF THE DISSERTATION Protein structure in reversible amyloid formed by low-complexity regions by Michael Patrick Hughes Doctor of Philosophy in Biological Chemistry University of California, Los Angeles, 2018 Professor David S. Eisenberg, Co-Chair Professor Kelsey C Martin, Co-Chair There is a current renaissance of research on membraneless organelles and their relationship to life in the cell. Protein-protein interactions between mysterious regions of proteins called Low Complexity Regions (LCRs) are known to be important for organizing these membraneless organelles. Membraneless organelles include: P-bodies, involved in mRNA degradation; the nucleolus, the center of rRNA genesis; Cajal bodies, related to telomerase function; and Stress Granules (SGs), dynamic organelles that form and disappear in response to stressful stimuli. LCRs from SG proteins form labile hydrogels composed of amyloid-like fibrils that are associated with organization of SGs. To elucidate the organization of these complexes we determined atomic resolution structures of adhesive five segments within LCRs. The resulting structures resemble known amyloid structures because the crystalized segments formed pairs of mating β-sheets that ran along a fibril axis. However, these five structures are distinct from known amyloid because of sharp kinks in the peptide backbones of the mating β-sheets. In conjunction with the relatively hydrophilic nature of these segments, the fibrils they form are labile; a departure from the stability associated ii with disease related amyloid. We termed this new type of structure low-complexity aromatic rich kinked segments (LARKS) and found their properties to be consistent with in vitro and cellular behavior needed to form dynamic membraneless assemblies found in the cell. A computational method, 3-D profiling, predicts that some 1725 proteins in the human proteome have two or more LARKS in LCRs that are consistent with properties needed to form membraneless assemblies. iii The dissertation of Michael Patrick Hughes is approved. Feng Guo Todd O Yeates Pascal Francois Egea David S. Eisenberg, Committee Co-Chair Kelsey C Martin, Committee Co-Chair University of California, Los Angeles 2018 iv Dedicated to my father for teaching me how the world works, and my mother for teaching me how to be a part of it. v TABLE OF CONTENTS List of Figures…………………………………………………………………………………………………………………………....vii List of Tables……………………………………………………………………………………………………………………………viii Acknowledgements…………………………………………………………………………………………………………………….ix Vita………………………………………………………………………………………………………………………………...………….xi Publications……………………………………………………………………………………………………………….…………......xii Overview ………………….…………………………………………………………………………………………………..1 Chapter 1 Discovery and characterization of low-complexity amyloid-like kinked segments in low-complexity regions…………….……………………………………….4 Chapter 2 Atomic structures of TDP-43 LCD segments and Insights into reversible or pathogenic aggregation…..…………………………………….33 Appendix I Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks (reprinted)……………….………….46 Appendix II Materials and methods………………………………………………...……………………………….78 References …………………………………………………………………………………...……………………………….81 vi LIST OF FIGURES Chapter 1: Figure 1.1. LCRs in FUS and hnRNPA1………………………………………..………..........................................………...5 Figure 1.2. Structure of LARKS…………………………………………….………………………………………………..….…7 Figure 1.3. Three-dimensional profiling to identify LARKS in LCRs of human proteins………………….8 Figure 1.4. Threading and 3D profiling on three separate LARKS structure backbones identify the same proteins as enriched in in LARKS……………….…………………...10 Figure 1.5. Threading identifies a unique set of LARKS-rich proteins….………………………………………12 Figure 1.6. Amino acid frequency in the human proteome comparted to the amino acid frequency for LCRs.……….……………………………………….………….…………..12 Figure 1.7. Qualitative categorization of the top 400 proteins rich in LARKS…….…………………………13 Figure 1.8. Sequence of Keratin 8………...….………………………………………………………………………...………14 Figure 1.9. Diagram of membraneless organelles found in human cells……...……………………...………..15 Figure 1.10. Histogram of the number of predicted LARKS in each protein in the human proteome.……...……………...……………...……………...……………………16 Figure 1.11. Origin of the 4.6 Å and absence of the 4.8 Å reflections…...…...………………………………….17 Figure 1.12. Powder diffraction patterns of the mCherry-FUS hydrogel and LARKS segments from Figure 2……………………………………………………………………..………17 Figure 1.13. LARKS construct forms a hydrogel……………..…………………………………………………………..18 Figure 1.14. A synthetic LARKS construct forms hydrogel with properties of the full length LCR of FUS……………………………………………………...……………………….19 Figure 1.15. Work flow for calculating area buried of segments in two mated sheets……..……………20 Figure 1.16. Solvation energies for LARKS and steric zippers……………………………………………………..21 Figure 1.17. Ramachandran plot of phi-psi angles found in LARKS structures…………….………………22 Figure 1.18. Aromatic side-chain stacking in steric zippers and LARKS…………………………..………….23 Figure 1.19. Hydrogen binding patterns in LARKS…………..…………..…………..…………..…………..………...24 Figure 1.20. Graphical model depicting mis-matched zippers…………………………………………………….25 vii Figure 1.21. Model of aromatic residues encouraging kinks in tight steric zippers...…………………….26 Figure 1.22. Size and variance of amino acids in LARKS and steric zippers.…………………………………27 Figure 1.23. LARKS crystal structure comparisons to cryoEM and ssNMR structures………….………28 Figure 1.24. Measuring kinkedness………………………………………………………………………………………...…29 Chapter 2 (reprinted): Figure 2.1. Segments from the LCD of TDP-43 form steric zippers……………………………………………...36 Figure 2.2. Validation of steric-zipper-forming segments………………………………………………..………….38 Figure 4.3. The NFGAFS segments form a kinked β-sheet that is strengthened by familial variants A315T and A315E……………………………………………...…………………...39 Figure 4.4. Speculative model showing the alternative pathways of formation of stress granules with pathogenic amyloid…………………………………………………………….40 LIST OF TABLES Chapter 1: Table 1.1. Fraction of residues in the human proteome found in LCRs, LARKS, or steric zippers…………………………………………………………….………………………..11 Table 1.2. GO terms enriched in the top 400 LARKS-rich proteins and their P-values……………………………..……………………………………………………….14 Chapter 2 (reprinted): Table 1.1. Data collection and refinement statistics…………………………………………………….….………….35 Table 1.2. MicroED data collection and refinement statistics……………………………………….….………….37 viii ACKNOWLEDGEMENTS This work was made possible entirely by the support and mentorship of my advisor, Professor David Eisenberg. David started me on this project, helped me make sense of each finding along the way, gave me the freedom and resources to pursue any question that interested me, and was tireless in editing manuscripts with me. I need to thank my committee who helped me navigate the world of science, and more importantly made the world of science a warm and welcoming place to learn. Thank you, Professors Kelsey Martin, Feng Guo, Pascal Egea, Todd Yeates, and David Eisenberg. Starting graduate school, I had no idea how to solve crystal structures and the technical aspects of structure determination were entirely alien to me. Duilio Cascio, Michael Sawaya, Meytal Landau, and Michael Collazo worked tirelessly and patiently with me, teaching me how to be a crystallographer and scientist. I would like to thank Tamir Gonen, Joze Rodriguez, and David Boyer for support with using MicroED. Without this tool available, many of my structures would remain a crystallographic attrition statistic. But instead they became atomic models. Past Eisenberg Lab alumna Elizabeth Guenter made substantial contributions to the LARKS project with her simultaneous discovery of kinked segments in the LCR of TDP-43. Elizabeth Guenther related these structures and disease mutations to stabilizing LARKS structures in vitro. This work greatly expanded the scope of the LARKS story and I am very appreciative of her contributions to this work. In addition, thank you to Dan Anderson and the Eisenberg lab community for helping to create an efficient lab space to work in. Special thanks to professors Steve McKnight, Roy Parker, and John-Paul Taylor for welcoming me into the membraneless organelle community and helping develop me as a scientist. Our collaborations were fantastic learning opportunities that I am lucky to have had. ix I worked with a very dedicated and talented undergraduate, Lisa Chong, on the nucleoporin portion of this work. Her help with crystallography made the scope of this project possible. I would like to acknowledge the Graduate Dean Scholar award, Cell and Molecular Biology training grant, Fowler Fellowship, and Dissertation year fellowship for financial support. I first learned lab work and scientific inquiry under Daniel Feliciano and Santiago Di Pietro. Both were very kind and supportive, ultimately leading to my studies at UCLA. Thank you to my family for their support in allowing me to pursue academics with reckless abandon. Especially S.W.H, L.P. and L.R. for helping me develop in my time in graduate