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Structure and Dynamics of Influenza M2 Proton Channels from Solid-State NMR By Structure and Dynamics of Influenza M2 Proton Channels from Solid-State NMR by Venkata Shiva Mandala B.A. Biochemistry Oberlin College, 2015 Submitted to the Department of Chemistry in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY February 2021 ©2020 Massachusetts Institute of Technology. All rights reserved. Signature of Author _____________________________________________________________ Department of Chemistry September 28, 2020 Certified by ____________________________________________________________________ Mei Hong Professor of Chemistry Thesis Supervisor Accepted by ___________________________________________________________________ Adam P. Willard Associate Professor of Chemistry Graduate Officer This doctoral thesis has been examined by a committee of professors from the Department of Chemistry as follows: ______________________________________________________________________________ Matthew D. Shoulders Whitehead Career Development Associate Professor Thesis Committee Chair ______________________________________________________________________________ Mei Hong Professor of Chemistry Thesis Supervisor ______________________________________________________________________________ Robert G. Griffin Arthur Amos Noyes Professor of Chemistry Thesis Committee Member 2 Structure and Dynamics of Influenza M2 Proton Channels from Solid-State NMR by Venkata Shiva Mandala Submitted to the Department of Chemistry on October 9, 2020 in Partial Fulfillment of the Requirements of the Degree of Doctor of Philosophy in Chemistry Abstract The A and B strains of influenza virus places a substantial burden on health, causing around 30 million illnesses, several hundred thousand hospitalizations, and a few tens of thousands of deaths every year in the United States alone. Developing novel antiviral and vaccines against influenza requires understanding the proteins employed by these infectious virions. The matrix-2 protein (M2) is an essential viral protein that conducts protons in the endosomes of infected host cells and induces membrane curvature to facilitate virus budding. M2’s proton channel activity is encapsulated by a transmembrane domain (TM) that is targeted by the FDA-approved antiviral drugs amantadine and rimantadine to inhibit viral replication. Proton conduction by M2’s TM is mediated by a His-xxx-Trp motif conserved in the otherwise disparate M2 from influenza A (AM2) and influenza B (BM2) strains. The histidine selects for protons and activates the channel at low pH, while the tryptophan is responsible for gating and unidirectional conduction from the N-terminus (outside) to the C-terminus (inside). M2 conducts protons across lipid bilayers at a moderate rate of ca. 10-1000 s-1. AM2 is well characterized and several high-resolution structures in a variety of membrane and membrane-mimetic environments are available, yet the mechanism of gating and the rate- limiting step of proton conduction are unknown. A gating-deficient mutant was utilized to determine that asymmetric conduction in AM2 is due to tryptophan blocking activation of histidine from the C-terminal side under low pH. Further, in phospholipid bilayers, AM2 shows two discrete conformations that interconvert on the proton conduction timescale, providing the first experimental evidence for a transporter-like mechanism. In contrast to AM2, BM2 is relatively poorly studied and no high-resolution structures in membranes is available. BM2 shares little sequence homology with AM2, is not inhibited by the antiviral drugs targeting influenza, and conducts protons faster but more bidirectionally than AM2. The first high-resolution structures of membrane-embedded BM2 in the closed and open states are reported. In contrast to the transporter-like motion of AM2, BM2 undergoes a subtler channel-like scissor opening motion, that allows for more efficient proton conduction at the expense of some bidirectional current. Thesis Supervisor: Mei Hong Title: Professor of Chemistry 3 Acknowledgments I first thank my research advisor Prof. Mei Hong, who gave me the opportunity to work on a wide variety of enriching and intriguing biological problems using solid-state NMR spectroscopy, and has been a constant source of support, encouragement, and fun scientific discussions during the last five years. Regardless of how busy she is, Mei’s door has always been open to teach NMR spectroscopy, bounce ideas, help troubleshoot problems in the lab, and generally be there during the good times and the difficult times. In addition to her presence, Mei has taught me to improve my scientific thought process, writing, figure-making and communication skills, both directly and through exposure to the wider scientific community. I am also very grateful to my committee members, Prof. Matthew Shoulders and Prof. Robert Griffin. Matt has been a constant source of encouragement and support over the last five years, and has provided keen insight into hypothesis-driven scientific approach, my fellowship applications, and the postdoctoral application process. I would like to thank Bob for his support, kind access to various scientific equipment, for hosting many interesting magnetic resonance seminars at the magnet lab, and for providing a solid quantum mechanics foundation to better understand NMR spectroscopy. I also thank my undergraduate honors research mentor, Prof. Manish Mehta, for his advice and motivation over the years. I have had the wonderful opportunity to collaborate with a number of talented researchers outside my lab. For the polyphosphate work, I thank Daniel Loh, Scott Shepard and Michael Geeson for all their hard work and informative discussions. Dr. Ivan Sergeyev at Bruker BioSpin was kind enough to collaborate for the DNP experiments in the polyphosphate study. Dr. Michiel Niesen and Dina Sharon’s expertise in molecular dynamics simulations was instrumental for completing the BM2 water study. I further thank Dr. Ivan Hung at the National High Magnet Field Laboratory for teaching me an efficient way to set up SPECIFIC-CP experiments. I express my gratitude towards past and present members of the Hong lab, for a very friendly and collaborative work environment. Dr. Shu-Yu Liao and Dr. Byungsu Kwon taught me the basics of NMR spectroscopy, molecular biology, and all the gory details of sample preparation, and were exceedingly patient and helpful in doing so. Their advice and collaboration was instrumental in completing the influenza AM2 work. Beyond direct contributions, Byungsu’s patience and dedication to all those around him in the lab was exemplary. I thank Marty Gelenter and Aurelio Dregni for their collaboration on various projects spanning M2, fibrils and methods, and countless discussions about NMR experiments. I also thank Marty for showing me his lifting routine at the gym, which was very helpful to a complete novice. I learnt a lot about fluorine NMR while working with Dr. Matthias Roos and Alex Shcherbakov. Alex was also kind enough to let me practice driving his car before getting my driver’s license. I also thank Dr. Myungwoon Lee, Dr. Tuo Wang, Dr. Jon Williams, and Dr. Hongwei Yao for helpful discussions and their continuous energy. Dr. Matt Elkins was an indispensable resource during my doctoral work and had advice on all sorts of scientific aspects and equipment troubleshooting. I learnt a lot from the diligent and patient work done by Dr. Matt McKay. I also have fond memories of my times getting lunch and interacting with Dr. Pyae Phyo, Dr. Pu Duan, Dr. Nhi Tran, Madeleine Sutherland, Sam Kaser, Harrison Wang, and all those mentioned above. 4 I also acknowledge the kind help from the technical, administrative and safety staff in the Magnet Lab and the Department of Chemistry, including Ajay Thakkar, Mike Mullins, Jennifer Weisman, Jay Matthews, Rebecca Teixeira, Wanda Hernandez, Jillian Haggerty, Karen Cote, Matt Fulton, and others. I am particularly thankful to Ajay and Mike, who were always exceedingly helpful and patient with troubleshooting probe and magnet technical issues. In addition to my lab mates, I enjoyed spending time with numerous other people during my five years at MIT. I had a lot of fun hanging out with Marty, Pyae, and others in the PChem cohort - Minjung Son, Dmitro Martynowych, Cole Perkinson, Crystal Chen, Kaitlyn Dwelle, Timothy Sinclair, Nicole Moody, and Jason Yoo, many of whom have graduated by now. I am glad I chanced upon being housemates with Minjung, who has been an amazing friend through graduate school. I thank Dr. Alex Loftis for the fun games of me getting beat at FIFA, but most importantly, his kind help and collaboration on the influenza BM2 project when I was stuck, which helped me get through the most trying time of my doctoral work. Travis Marshall-Roth has been an unbelievable flatmate and friend during my time here as well, and I thank him for many enjoyable bike rides over the years. Finally, I thank the numerous groups and people I have run with or played soccer with over the years at MIT, including Abhishek, Tony, Rohit, the PSFC, SDM, Chemistry and Hope teams, the random Soccer@MIT whatsapp groups, and more recently, the Boston Soccer group. Soccer and running have provided a constant source of enjoyment in my life and helped keep me in good spirits, especially through the challenging times. I also thank my friends from college and elsewhere, including Holden, Thomas, Elana, Prakash, Daniella, Pablo, Charlotte, Rand, Sakina, Hind, Ted, Eric, and
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