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STRUCTURAL AND FUNCTIONAL STUDIES OF TRPML1 AND TRPP2 Nicole Marie Benvin Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2017 © 2017 Nicole Marie Benvin All Rights Reserved ABSTRACT Structural and Functional Studies of TRPML1 and TRPP2 Nicole Marie Benvin In recent years, the determination of several high-resolution structures of transient receptor potential (TRP) channels has led to significant progress within this field. The primary focus of this dissertation is to elucidate the structural characterization of TRPML1 and TRPP2. Mutations in TRPML1 cause mucolipidosis type IV (MLIV), a rare neurodegenerative lysosomal storage disorder. We determined the first high-resolution crystal structures of the human TRPML1 I-II linker domain using X-ray crystallography at pH 4.5, pH 6.0, and pH 7.5. These structures revealed a tetramer with a highly electronegative central pore which plays a role in the dual Ca2+/pH regulation of TRPML1. Notably, these physiologically relevant structures of the I-II linker domain harbor three MLIV-causing mutations. Our findings suggest that these pathogenic mutations destabilize not only the tetrameric structure of the I-II linker, but also the overall architecture of full-length TRPML1. In addition, TRPML1 proteins containing MLIV- causing mutations mislocalized in the cell when imaged by confocal fluorescence microscopy. Mutations in TRPP2 cause autosomal dominant polycystic kidney disease (ADPKD). Since novel technological advances in single-particle cryo-electron microscopy have now enabled the determination of high-resolution membrane protein structures, we set out to solve the structure of TRPP2 using this technique. Our investigations offer valuable insight into the optimization of TRPP2 protein purification and sample preparation procedures necessary for structural analysis. Table of Contents List of Figures and Tables ........................................................................................................... vi Acknowledgements ...................................................................................................................... ix Dedication .................................................................................................................................... xii Chapter I Introduction to TRPML1 and TRPP2 1.1 Transient receptor potential (TRP) channel family .................................................................. 1 1.2 Introduction to the TRPML1 protein ........................................................................................ 3 1.2.1 TRPML1 and its role in mucolipidosis type IV (MLIV) ................................................... 4 1.2.2 Subcellular localization and channel regulation of TRPML1 ........................................... 4 1.2.3 Trafficking of TRPML1 ..................................................................................................... 6 1.2.4 The TRPML subfamily ..................................................................................................... 7 1.2.5 Electrophysiological properties of TRPML channels ........................................................ 7 1.3 Introduction to the TRPP2 protein and ADPKD ...................................................................... 9 1.3.1 Clinical manifestations of ADPKD .................................................................................. 10 1.3.2 Pathogenesis of ADPKD .................................................................................................. 11 1.3.3 Cellular and subcellular localization of TRPP2 ............................................................... 12 1.3.4 Mechanotransduction model of the PKD1/TRPP2 complex ............................................ 13 i 1.3.5 TRPP2 and its role in ADPKD ......................................................................................... 14 1.3.6 Stoichiometric assembly of the PKD1/TRPP2 complex .................................................. 14 1.3.7 TRPP2 agonist triptolide .................................................................................................. 15 1.3.8 Interaction between TRPP2 and additional channels ....................................................... 16 1.3.9 Ion selectivity in TRPP2 .................................................................................................. 16 1.3.10 Additional members of the TRPP subfamily ................................................................. 17 1.4 Structural determination of TRP channels .............................................................................. 18 1.5 Conclusion and thesis overview ............................................................................................. 20 1.6 References ............................................................................................................................... 22 Chapter II Structural and Functional Studies of the TRPML1 I-II Linker 2.1 Introduction ............................................................................................................................. 30 2.2 Experimental procedures for structrual studies ...................................................................... 31 2.2.1 Protein expression and purification of the TRPML1 I-II linker ...................................... 31 2.2.2 Protein crystallization of the TRPML1 I-II linker ........................................................... 33 2.2.3 Data collection and structure determination .................................................................... 34 2.3 Structural features of the TRPML1 I-II linker structures ....................................................... 38 2.3.1 TRPML1 I-II linker structure at pH 6.0 ........................................................................... 38 2.3.2 Features of the central luminal pore ................................................................................ 39 2.3.3 TRPML1 I-II linker structures at pH 4.5 and pH 7.5 ....................................................... 41 ii 2.3.4 Verification of the tetrameric I-II linker .......................................................................... 43 2.3.5 Role of the I-II linker in TRPML1 assembly .................................................................. 45 2.4 Structural basis of pH and Ca2+ regulation of TRPML1......................................................... 47 2.4.1 Dual regulation of TRPML1 activity by pH and Ca2+ ..................................................... 47 2.4.2 The central pore is essential for pH and Ca2+ regulation .................................................. 48 2.4.3 Model of pH and Ca2+ dual regulation of TRPML1 ......................................................... 50 2.5 Characterization of MLIV-causing mutations ........................................................................ 53 2.5.1 MLIV-causing mutations in the TRPML1 I-II linker ...................................................... 53 2.5.2 Cell culture, transfection, and confocal live-cell imaging ............................................... 54 2.5.3 Confocal imaging of MLIV-causing mutations .............................................................. 55 2.5.4 Biochemical analysis and circular dichroism spectroscopy ............................................ 58 2.6 Conclusion .............................................................................................................................. 60 2.7 References .............................................................................................................................. 62 Chapter III Structural Studies of TRPP2 3.1 Introduction ............................................................................................................................. 64 3.2 Screening of TRPP2 constructs using FSEC ......................................................................... 65 3.2.1 Rationale for cloning truncated constructs ....................................................................... 65 3.2.2 Transient HEK cell transfection and FSEC...................................................................... 66 3.2.3 FSEC analysis of human TRPP2 constructs transfected in HEK293 cells ..................... 67 3.3 Insect cell baculovirus-based expression system .................................................................... 69 iii 3.3.1 Generation of recombinant baculoviruses ........................................................................ 69 3.3.2 FSEC analysis of human TRPP2 constructs transfected into Sf9 cells ............................ 70 3.4 FSEC of mouse TRPP2 constructs transfected in HEK293 cells ........................................... 71 3.5 Purification of hTRPP2 in Hi5 cells using DDM and amphipol ............................................ 73 3.5.1 Small-scale purification in DDM ..................................................................................... 74 3.5.2 Reconstitution into amphipols .......................................................................................... 75 3.5.3 Inhibition of N-glycosylation ........................................................................................... 79 3.5.4 Large-scale purification of TRPP2 in DDM ................................................................... 82 3.6 Purification of hTRPP2 in Hi5 cells using LMNG ................................................................
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