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Mechanism of Phospholamban Activation by HAX-1 and Their Roles in the Regulation of the SR Calcium-Atpase

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Mechanism of Phospholamban Activation by HAX-1 and Their Roles in the Regulation of the SR Calcium-Atpase Mechanism of Phospholamban Activation by HAX-1 and their Roles in the Regulation of the SR Calcium-ATPase A Dissertation SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Erik Keith Larsen IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Advisor Dr. Jiali Gao April 2021 © Erik Keith Larsen 2021 Abstract The Ca2+ transient of the cardiomyocyte is key to the contractility of the heart. Its dysregulation has been associated with heart disease, leading to investigation of the regulation of Ca2+ for potential drug targets. The Sarcoplasmic Endo-Reticulum Ca2+ ATP-ase (SERCA) pump and its main inhibitor in heart tissue, phospholamban (PLN), are two promising targets that are under β-adrenergic control via phosphorylation of PLN by Protein Kinase A (PKA). Phosphorylation of Ser16 on the cytoplasmic domain of PLN results in decreased inhibition of SERCA. Recently, an additional member of the SERCA interactome has been discovered called Hematopoietic lineage cell-specific protein 1 (HCLS1) Associated Protein X-1 (HAX-1). Contrasting PLN phosphorylation, the interaction of HAX-1 and PLN increases the inhibition of PLN for SERCA, adding another layer of complexity to SERCA regulation and a potential new drug target. This thesis aims to investigate the structure-function relationship of the ternary complex, SERCA/PLN/HAX-1 using NMR spectroscopy as the primary technique. i Table of Contents Abstract ......................................................................................................................... i List of Tables .............................................................................................................. vi List of Figures ............................................................................................................ vii List of Abbreviations .................................................................................................. ix Chapter 1 Introduction .................................................................................................... 1 1.1 Entropy and information ....................................................................................... 1 1.2 Lipid membranes ................................................................................................... 3 1.3 Membrane proteins ............................................................................................... 4 1.4 Integral membrane proteins ................................................................................. 6 1.5 Peripheral membrane proteins ............................................................................. 7 1.6 Intrinsically disordered proteins ........................................................................ 10 1.7 Cardiophysiology ................................................................................................ 14 1.8 Therapeutic targets for heart failure .................................................................. 17 1.9 Objectives ............................................................................................................ 22 1.10 NMR spectroscopy ............................................................................................ 23 1.10.1 Basics of NMR .............................................................................................. 24 1.10.2 Chemical shift ............................................................................................... 32 1.10.3 Direct dipole-dipole coupling ......................................................................... 34 1.10.4 J-coupling ...................................................................................................... 35 1.10.5 Spin rotation .................................................................................................. 35 1.10.6 Signal detection ............................................................................................ 36 1.10.7 Relaxation ..................................................................................................... 38 1.10.8 Solid state NMR ............................................................................................ 41 Chapter 2 Solid-state NMR of membrane proteins in lipid bilayers: to spin or not to spin? ............................................................................................................................... 45 2.1 Introduction .......................................................................................................... 48 2.2 Membrane mimetic systems for high-resolution ssNMR spectroscopy ........ 50 2.3 To spin or not to spin? ........................................................................................ 52 2.4 Advanced MAS and OS-ssNMR techniques ...................................................... 54 2.5 Structure calculations using ssNMR restraints ................................................ 58 2.6 Topological allostery: transmembrane signaling via dynamic interactions .. 60 2.7 Characterization of motions via ssNMR ............................................................ 63 ii 2.8 Interactions of intrinsically disordered proteins (IDPs) with lipid membranes ..................................................................................................................................... 65 2.9 Conclusions and perspectives ........................................................................... 66 2.10 Biographical information .................................................................................. 67 Chapter 3 Probing protein-protein interactions using asymmetric labeling and carbonyl-carbon selective heteronuclear NMR spectroscopy .................................. 70 3.1 Introduction .......................................................................................................... 72 3.2 Results .................................................................................................................. 73 3.2.1 Mapping two binding partners fingerprints simultaneously ............................. 73 3.2.2 Fingerprinting three binding partners using one sample ................................. 77 3.2.3 Measuring Residual Dipolar Coupling (RDC) of complexes using one sample ................................................................................................................................. 80 3.2.4 Measuring long-range distances and transient complexes using CCLS for Paramagnetic Relaxation Enhancements (PRE) ..................................................... 82 3.2.5 Improving sensitivity with the G5 pulse ........................................................... 85 3.3 Conclusion and perspectives ............................................................................. 86 3.4 Acknowledgements ............................................................................................. 87 Chapter 4 Cysteine-ethylation of tissue-extracted membrane proteins as a tool to detect conformational states by solid-state NMR spectroscopy .............................. 88 4.1 Introduction .......................................................................................................... 90 4.2 Protocols for using 13C-EMTS ............................................................................ 92 4.2.1 Synthesis and purification of the 13C-EMTS reagent ....................................... 92 4.2.2 Ethylation and reconstitution of membrane proteins into lipid bilayers ........... 98 4.2.3 Rotor packing ................................................................................................ 100 4.3 Solid-state NMR spectroscopy ......................................................................... 102 4.3.1 Hardware requirements ................................................................................ 102 4.3.2 Cross-polarization and general considerations ............................................. 103 4.3.3 Sample temperature ..................................................................................... 105 4.3.4 Optimization and acquisition of [13C,13C]-DARR spectra ............................... 106 4.3.5 Assignment of labeled cysteins ..................................................................... 109 4.4. Conclusions and perspectives ........................................................................ 110 4.5 Acknowledgments ............................................................................................. 113 Chapter 5 Structural basis for allosteric control of the SERCA-phospholamban membrane complex by Ca2+ and phosphorylation................................................... 114 5.1 Introduction ........................................................................................................ 115 iii 5.2 .Results ............................................................................................................... 118 5.2.1 The TM domain of PLN undergoes a topological two-state equilibrium ........ 118 5.2.2 PLN phosphorylation by PKA signals a rearrangement of the SERCA/PLN binding interface ..................................................................................................... 121 5.2.3 Dynamic structural refinement of the SERCA-PLN complexes ..................... 123 5.2.4 Phosphorylation disrupts correlated motions between PLNAFA and SERCA’s Ca2+ binding sites ................................................................................................... 129 5.2.5 Effects of Ca2+
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