GIST Repository
College of Life Sciences and Medical Engineering
Department of Life Sciences
4. Theses(Ph.D)
Structural and functional study of transporters: ABCB6 and GLUT7 by using cryo-EM
- Abstract
- Over the past decade, remarkable advancements in cryo-electron microscopy (cryo-EM) have
revolutionized the field of structural biology. Among various innovations, my focus has been on the structural analysis of membrane proteins. Due to their intrinsic characteristics, such as requiring diverse membrane reconstitution methods during the purification process, membrane proteins have posed significant challenges in structure determination using X-ray crystallography, which relies on obtaining high-quality crystals for diffraction pattern analysis. However, with the development of cryo-EM, a new avenue has emerged for determining membrane protein structures. Leveraging these advancements, I conducted structural studies on membrane proteins, particularly transporters. In the first part of this study, I targeted ATP Binding Cassette (ABC) transporters, which hydrolyze ATP and exhibit a broad substrate spectrum. Specifically, I determined the structure of ABCB6, the sixth protein in the B family of ABC transporters. Despite ABCB6 being relatively well-studied, I elucidated the previously unresolved transition state, the outward-facing open conformation, at a resolution of 3.8 Å. Additionally, I revealed that lipids play a structural role in stabilizing the protein. In the second part, I determined the structure of glucose transporter 7 (GLUT7), a protein known to transport glucose, one of the most fundamental energy sources in biological systems. Given its small molecular weight of 55 kDa, structure determination using cryo-EM was considered challenging. However, by employing nanodiscs and implementing a reference-based filtering process during data processing to eliminate bad particles, I achieved a final resolution of 3.3 Å. This study provides insights into the structural analysis of membrane proteins and the characterization of protein transition states using cryo-EM. The newly determined structures serve as foundational data for future research in this field.
- Issued Date
- 2025
- Type
- Thesis
- Alternative Author(s)
- Sang Soo, Lee
- Department
- 대학원 생명과학부
- Advisor
- Jin, Mi Sun
- Table Of Contents
- by employing nanodiscs and implementing a reference-based filtering process during data processing to
eliminate bad particles, I achieved a final resoluti n of 3.3 Å.
This study provides insights into the structural analysis of membrane proteins and the characterization
of protein transition states using cryo-EM. The newly determined structures serve as foundational data for
future research in this field.
CONTENTS
PART I
ABSTRACTS i
CONTENTS iii
LIST OF TABLES AND FIGURES iv
I. INTRODUCTION 1
II. MATERIALS AND METHODS
2.1. Cloning, expression and purification of ABCB6 and its mutants 3
2.2. Cloning, expression and purification of hApoA-I ((Δ1−43) 4
2.3. Nanodisc reconstitution 4
2.4. ATP hydrolysis assay 5
2.5. Proteoliposome reconstitution 5
2.6. Proteoliposome transport activity assay for CPIII 6
2.7. CryoEM grid preparation and data collection 6
2.8 CryoEM data processing 6
2.9 Model building and refinement 7
III. RESULTS
3.1. VO4 and AlF4 can inhibit the catalytic cycle, capturing the post-ATP hydrolysis state 8
3.2. An application to prefer the outward-facing conformation in hABCB6core 8
3.3. The impact of the W546A mutation on the ATPase and transport functions of hABCB6core 9
3.4. The W546A mutant favors the outward-facing state in detergent environment but not in nanodisc
reconstitution · · · · · · · · · · · · · ·· · · · · · · · · · · · · · · · · ·· · · · · · · · ·· · · · · · · · · · · · · · · · · ·· · · · · · · 10
3.5. Overall structure of outward-facing conformation 12
3.6. The outward-facing conformation has low substrate affinity, facilitating its release 12
IV. DISCUSSION 14
LIST OF TABLES AND FIGURES
Figure 1. Inhibition of the ATPase activity of hABC6core by ATP analogs 16
Figure 2. Purification profile of hABCB6coreand proteoliposome 18
Figure 3. Representative cryoEM data processing results 20
Figure 4. Structural difference depending on conformation 22
Figure 5. Well-conserved the central interactions through the substrate pathway ·· 24
Figure 6. ATPase activity of ABCB6core and W546A proteins 26
Figure 7. Purification profile of W546A mutant· 28
Figure 8. Comparison of the hABCB6core - W546A mutant in detergent and Nanodisc 30
Figure 9. Electron density of lipids interacting with TMDs hydrophobic region 32
Figure 10. The post-occluded conformation of the hABCB6core-W546A mutant is
stabilized by a phospholipid embedded within the hydrophobic groove of the
TMD surface 34
Figure 11. The structural overview of the hABCB6core-W546A mutant in an outward-
facing conformation· 36
Figure 12. During the CPIII transport cycle, assumed conformational change on the
substrate binding site with all structure data of ABCB6 38
Figure 13. During the Hemin:GSH transport cycle, assumed conformational change on
the substrate binding site with all structure data of ABCB6· 40
Figure 14. Cryo-EM data processing pipeline employed for detergent-purified
hABCB6core in the presence of CPIII and Mg2+/ADP·VO4 42
Figure 15. Cryo-EM data processing pipeline employed for Mg2+/ADP·VO4-bound,
outward-facing hABCB6core-W546A in detergent micelles 44
Figure 16. Cryo-EM maps of outward-facing hABCB6core-W546A in complex with
Mg2+
/ADP·VO4 46
Figure 17. Cryo-EM data quality analysis of outward-facing hABC 6core-W546A in
complex with Mg2+/ADP·VO4· 48
Figure 18. Cryo-EM data processing pipeline employed for Mg2+/ADP·VO4-bound, post-
occluded hABCB6core-W546A in nanodiscs 50
Figure 19. Cryo-EM maps of the post-occluded hABCB6core-W546A in complex with
Mg2+
/ADP·VO4 52
Figure 20. Cryo-EM data quality analysis of the post-occluded hABCB6core-W546A in
complex with Mg2+
/ADP·VO4 54
Figure 21. Schematic diagrams of conformational equilibria between the outward-facing
and post-occluded configurations of hABCB6core and the W546A mutant
56
Figure 22. Role of conserved aromatic residues in stabilizing the occluded conformation
of the ABC transporter family 58
Table 1. Cryo-EM data collection, refinement and validation statistics 60
PART II
ABSTRACTS i
CONTENTS iii
LIST OF TABLES AND FIGURES iv
I. INTRODUCTION 62
II. MATERIALS AND METHODS
2.1. Cloning, expression and purification 63
2.2. Nanodisc reconstitution 64
2.3. Grid preparation and data acquisition 64
2.4. Electron microscopy data processing 64
2.5. Model building and refinement 65
III. RESULTS AND DICUSSION
3.1. Structure detemination·· ·· · ·· · · ·· · ·· · ·· · ·· · ·· · · ·· · ·· · · ·· · ·· · · ·· · ·· · ·· · ·· · ·· · ·· · · ·· 66
3.2. Overall fold· · · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · · · · · · · ·· · · · ·· · · · · · · · · · ·· · · · · · · · · ·· · · · · 67
3.3. Substrate-binding cavity· 67
3.4. Alternating access cycle· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · · · 68
LIST OF TABLES AND FIGURES
Figure 1. Cryo-EM image and processing flowchart· 70
Figure 2. The quality of Cryo-EM processing results 72
Figure 3. Topology of human GLUT7 74
Figure 4. Outward-open structure of human GLUT7 76
Figure 5. Substrate-binding cavity 78
Figure 6. Possible mechanism of the conformational transition 80
Table 1. Cryo-EM data collection, refinement and validation statistics 82
ABSTRACT IN KOREAN 85
ACKNOWLEDGEMENT 86
REFERENCES 87
- Degree
- Doctor
- Appears in Collections:
- Department of Life Sciences > 4. Theses(Ph.D)
- 파일 목록
-
-
관련 파일이 존재하지 않습니다.
-
Items in Repository are protected by copyright, with all rights reserved, unless otherwise indicated.