Title Page
ABSTRACT
Contents
CHAPTER I. INTRODUCTION 11
1.1. Background 12
1.1.1. Structural study of G protein coupled receptor 12
1.1.2. Neuropeptide Y and its receptor, NPYR 13
1.2. Purpose of this study 21
1.2.1. Limitation of previous research 21
1.2.2. Significance and application 21
CHAPTER II. MATERIAL AND METHODS 23
2.1. Structure determination of NPY-Y₁R-G protein complex 24
2.1.1. Purification of Y₁R 24
2.1.2. Purification of G protein heterotrimer 25
2.1.3. Purification of scFv16 26
2.1.4. Complex formation and purification 26
2.1.5. Cryo-EM grid preparation and data collection 27
2.1.6. Cryo-EM data processing 27
2.1.7. Model building and refinement 28
2.2. Structure determination of NPY/PYY(3-36)-Y₂R-G protein complexes 28
2.2.1. Purification of Y₂R 28
2.2.2. Purification of G protein heterotrimer 29
2.2.3. Purification of ScFv16 30
2.2.4. Complex formation and purification 30
2.2.5. Cryo-EM grid preparation and data collection 31
2.2.6. Cryo-EM data processing 31
2.2.7. Model building and refinement 33
2.3. Functional assays 34
2.3.1. ELISA-based surface expression assay 34
2.3.2. Ca2+ signaling assay[이미지참조] 35
CHAPTER III. RESULTS 44
3.1. Structural and functional analysis on Y₁R complex 45
3.1.1. Overall structure of Y₁R complex 45
3.1.2. Binding of the C-terminus of NPY to Y₁R 51
3.1.3. Binding of the N-terminal and helical regions of NPY 61
3.2. Structural and functional analysis on Y₂R complexes 71
3.2.1. Overall structure of Y₂R complexes 71
3.2.2. Binding of the C-terminus of NPY/PYY(3-36) to Y₂R 82
3.2.3. Binding of the N-terminal and helical regions of NPY/PYY(3-36) 88
3.3. Comparison between Y₁R and Y₂R 94
CHAPTER IV. CONCLUSION 101
REFERENCES 110
Appendix 123
초록 125
Table 1. Cryo-EM data collection and processing parameters. 37
Table 2. Cryo-EM structure refinement statistics. 38
Table 3. Ca2+ signaling assays of wild-type and mutant Y₁ receptors.[이미지참조] 55
Table 4. Ca2+ signaling assays of wild-type Y₁ receptor with different peptide ligands.[이미지참조] 64
Table 5. Ca2+ signaling assays of wild-type Y₂ receptor with different peptide ligands.[이미지참조] 74
Table 6. Ca2+ signaling assays of wild-type and mutant Y₂ receptors.[이미지참조] 75
Figure 1. Schematic view of G protein-coupled receptor signaling. 16
Figure 2. Common activation model for class A GPCRs. 17
Figure 3. Neuropeptide Y (NPY) and NPY receptor (NPYR) expression in CNS. 18
Figure 4. Sequene alignment between NPY family peptides. 19
Figure 5. Receptor subtype preference of NPY family peptides. 20
Figure 6. Sample preparation and cryo-EM data analysis (Y₁R). 39
Figure 7. Sample preparation and cryo-EM data analysis (Y₂R). 41
Figure 8. Surface expression levels of Y₁R/Y₂R mutants. 43
Figure 9. Overall structure of the NPY-Y1R-Gi-scFv16 complex.[이미지참조] 47
Figure 10. Comparison between NPY-and antagonist-bound Y₁R structures. 48
Figure 11. Gi binding interface.[이미지참조] 50
Figure 12. Binding of NPY C-terminal tail to Y₁R. 56
Figure 13. Ca2+ signaling assays (Y₁R mutants related to binding of NPY C-terminal).[이미지참조][이미지참조] 57
Figure 14. Intramolecular interaction between R35 and Y36 of NPY. 58
Figure 15. Comparison of Y₁R binding mode between NPY and antagonists. 59
Figure 16. Binding of NPY N-terminus and helical region to Y₁R. 65
Figure 17. Ca2+ signaling assays (Y₁R mutants related to binding of NPY N-terminus and helical region).[이미지참조] 66
Figure 18. F202 contributes to the structural integrity of Y₁R. 67
Figure 19. Cryo-EM density of NPY peptide at different threshold 68
Figure 20. Continuous density of the N-terminal region of Y₁R beyond TM1. 69
Figure 21. Ca2+ signaling assays (N-terminal mutants of Y₁R).[이미지참조] 70
Figure 22. Downstream signaling of Y₂R with NPY/PYY and their analogs. 76
Figure 23. Ca2+ signaling assays of wild-type Y₂ receptor and H1493.51Y/S2806.47C mutant[이미지참조] 77
Figure 24. Overall structures of NPY/PYY(3-36)-Y₂R-Gi-scFv16 complexes and structural comparison between inactive and active Y₂R.[이미지참조] 78
Figure 25. Conformational changes of Y₂R by activation with NPY/PYY(3‒36). 80
Figure 26. NPY/PYY(3-36) binding at the transmembrane domain of Y₂R. 84
Figure 27. Downstream signaling of Y₂R with NPY and Y36 mutants. 86
Figure 28. Ca2+ signaling assays of wild-type Y₂ receptor and mutants (Y₂R mutants related to binding of NPY/PYY(3-36) C-terminus).[이미지참조] 87
Figure 29. NPY/PYY(3-36) binding at the extracellular region of Y₂R. 90
Figure 30. Ca2+ signaling assays of wild-type Y₂ receptor and mutants (Y₂R mutants related to binding of NPY/PYY(3-36) helical region).[이미지참조] 92
Figure 31. Ca2+ signaling assays of wild-type Y₂ receptor and mutants (Y₂R ECL12 mutants).[이미지참조] 93
Figure 32. Structure comparison between NPY-bound Y₁R and Y₂R. 97
Figure 33. Ca2+ signaling assays of wild-type Y₂ receptor and mutant (V2916.58F).[이미지참조] 99
Figure 34. NPY-Y₁R and NPY-Y₂R structures show different interaction pattern at ECL2 region of receptors. 100
Figure 35. Mechanism of Y₁R activation by NPY binding. 107
Figure 36. Alignment with recently published NPY bound Y₂R. 108
Figure 37. Schematic representation of NPY binding modes in Y₁R and Y₂R. 109
Appendix Figure 1. Published paper. 124