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Insight Into Ca2+ Dependent Lipid Scrambling from the Crystal Structure of a TMEM16 Family Member Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2015 Insight into Ca2+ Dependent Lipid Scrambling from the Crystal Structure of a TMEM16 Family Member Brunner, Janine Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-113153 Dissertation Published Version Originally published at: Brunner, Janine. Insight into Ca2+ Dependent Lipid Scrambling from the Crystal Structure of a TMEM16 Family Member. 2015, University of Zurich, Faculty of Science. Insight into Ca2+ Dependent Lipid Scrambling from the Crystal Structure of a TMEM16 Family Member Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Janine Denise Brunner von Ebnat-Kappel SG Promotionskomitee Prof. Dr. Raimund Dutzler (Leitung und Vorsitz) Prof. Dr. Markus Grütter Prof. Dr. Markus Seeger Zürich, 2015 Dedicated to my mother Yolanda Brunner 14. 06. 1956 - 16. 04. 2006 Acknowledgements First, I would like to express my gratitude to Prof. Raimund Dutzler for giving me the opportunity to work on that great project on TMEM16 proteins. I am thankful to the members of my thesis committee for their interest, Prof. Markus Gruetter and Prof. Markus Seeger. I would like to thank Alessia for her appreciated help during her master thesis, also for constant support with cupcakes. I am grateful to Stephan for a great time in the past five years, inside as well as outside of the lab, and for support throughout my PhD, for invaluable discussions on scientific topics while drinking our daily coffee and for introduction to the HEK expression system. I appreciated a lot our teamwork on shared projects. I would like to thank Sibylle for many funny hours throughout my PhD and for valued chats, making L24 a nice place. I would like to express my gratitude to Nita, always amiable, and usually finding each other working late in the lab, for mental support. I would like to thank Novandy for introduction to the yeast expression system. I would like to thank Yvonne for constant effort to organize the lab and make it a pleasant place. I wish to express my gratitude to former members of the Gruetter lab and to the present members of the Jinek lab, who provided a good atmosphere inside and outside the lab, and who made the L-floor a pleasant place. Special thank goes to Michi, for his constant support and friendship in good times and in bad, Heidi, Georg and Cristopher for introduction to the insect cell expression system, for helpful discussions and valued chats. Also, I appreciated the introduction to the art of brewery through Cristopher. I want to say thanks to the infrastructure of the Department of Biochemistry, especially Sascha for designing and constructing the HPL6 cell lyser, which made membrane preps a comfortable thing and for valued chats. I would like to express my gratitude to Sascha, Adi and Lukas from the workshop in general for support, and to Beat and Celine from the protein crystallization facility for constant support and valued chats, and to the IT team, Stefan, Steve and Jannik, for solving all kinds of computer problems. I wish to express my deepest gratitude to my friends and family for endless support throughout the past years. I List of Figures Figure 1 Phospholipids and cholesterol ..................................................................................... 3 Figure 2 Membrane asymmetry in a eukaryotic cell ................................................................. 4 Figure 3 Events during blood clotting ....................................................................................... 6 Figure 4 Maintenance and breakdown of the lipid asymmetry at the plasma membrane ......... 9 Figure 5 Alignment of murine TMEM16A with TMEM16F .................................................. 15 Figure 6 Predicted topology of TMEM16 proteins ................................................................. 17 Figure 7 Block to polyspermy in Xenopus laevis oocytes after fertilization ........................... 20 Figure 8 ER- PM tether by Ist2 in Saccharomyces cerevisiae ................................................ 22 Figure 9 Origin and sequence length of the TMEM16 homologues ....................................... 28 Figure 10 Relationship between selected TMEM16 members from vertebrates..................... 29 Figure 11 Relationship between fungal and murine TMEM16 proteins ................................. 30 Figure 12 Sequence alignment of the TM part of murine TMEM16 proteins ......................... 31 Figure 13 Secondary structure elements in the N- terminus.................................................... 32 Figure 14 Sequence alignment of the N- termini of TMEM16 proteins ................................. 33 Figure 15 Sequence alignment of the loop regions of TMEM16 members ............................ 34 Figure 16 Circular phylogramm of all TMEM16 homologues used in this work ................... 36 Figure 17 Compartment localization of TMEM16 family members in HEK cells ................. 39 Figure 18 Expression screening (HEK cells) evaluated by FSEC (part a) .............................. 40 Figure 19 Expression screening (HEK cells) evaluated by FSEC (part b) .............................. 41 Figure 20 Small-scale purification of gzeTMEM16 expressed in insect cells ........................ 43 Figure 21 Expression screening (S. cerevisiae) evaluated by FSEC ....................................... 44 Figure 22 Medium scale purifications from HEK cell expression (a) ..................................... 45 Figure 23 Medium scale purifications from HEK cell expression (b) ..................................... 46 Figure 24 Medium scale purifications from S. cerevisiae expression (a) ................................ 47 Figure 25 Medium scale purifications from S. cerevisiae expression (b) ............................... 48 Figure 26 Size exclusion of a large scale purification of ppaTMEM16 from HEK cell culture ................................................................................................................................................. 49 Figure 27 Size exclusion chromatography of a large scale purification of gzeTMEM16 from HEK cell culture ...................................................................................................................... 50 Figure 28 Diffraction of gzeTMEM16 crystals ....................................................................... 51 Figure 29 Gel filtration chromatograms of gzeTMEM16 purified from insect cell culture .... 52 Figure 30 Thermal stability assay with gzeTMEM16 (raw data) ............................................ 54 Figure 31 Thermal stability assay with gzeTMEM16 ............................................................. 55 Figure 32 Stabilized vs. not stabilized gzeTMEM16 from HEK cells .................................... 56 Figure 33 Stabilized versus not stabilized gzeTMEM16 from S.cerevisieae .......................... 57 Figure 34 Thermal stability of nhTMEM16 ............................................................................ 59 Figure 35 Gel filtration profile of nhTMEM16 ....................................................................... 60 Figure 36 nhTMEM16 crystals and diffraction pattern ........................................................... 63 Figure 37 From BLAST search to high-resolution crystals: workflow ................................... 65 Figure 38 Methionine synthesis in Saccharomyces cerevisiae................................................ 67 Figure 39 Optimizing expression conditions of Se-Met derivatized nhTMEM16 .................. 68 Figure 40 Gel filtration profile of Se-Met labelled nhTMEM16 ............................................. 69 Figure 41 Se-Met incorporation revealed by mass spectrometric analysis ............................. 70 Figure 42 Statistics from SHELXC ......................................................................................... 71 II Figure 43 Solutions obtained with SHELXD .......................................................................... 72 Figure 44 Statistical output of SHELXD: Selenium site occupancy ....................................... 72 Figure 45 Density modification by SHELXE.......................................................................... 73 Figure 46 Electron density of the refined model ..................................................................... 74 Figure 47 Anomalous difference density map of data collected from Met mutants ................ 75 Figure 48 Ca2+ binding site ...................................................................................................... 76 Figure 49 Statistics on data collected in the apparent absence of Ca2+ ................................... 76 Figure 50 Electron density in the ‘Ca2+-free’ structure ........................................................... 77 Figure 51 The structure of nhTMEM16 .................................................................................. 78 Figure 52 Features of the amino- and carboxy termini ........................................................... 78 Figure 53 Mutual interaction between conserved glutamates and histidines .......................... 79 Figure 54 The Ca2+ binding site............................................................................................... 80 Figure 55 Subunit- cavity .......................................................................................................
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