臺灣博碩士論文加值系統

PML protein is mainly present inside the nucleus in the form of PML nuclear bodies (PML-NBs) and serves as a SUMO E3 ligase which facilitates SUMOylation on PML itself and proteins in PML-NBs. PML has a conserved N-terminal TRIM/RBCC region comprised of a RING finger domain, two B-boxes and a coiled-coil region. The purpose of this study is to investigate the structural basis of SUMOylation on PML TRIM domains which is essential in the formation of PML-NBs. However, not much of the structural information of PML is known today. In this work we solved the structures of RING finger and B-box 1 of dimer by advanced NMR techniques. We identified the residues involved in the binding of SUMO E2 enzyme Ubc9 and PML TRIM domains. The result of SPR indicated that dimeric domain RB1 showed much stronger interaction with Ubc9 (KD=16uM) than any single domain (KD=490uM for RING finger and KD=94uM for B-box 1), suggesting that RING finger and B-box 1 bound with Ubc9 in synergy. Here, we provide the structures of PML TRIM domains and the bindings with Ubc9 at molecular level in order to understand the SUMOylation mechanism on PML TRIM domains.

Table of Contents

Verification letter from the Oral Examination Committee i
Acknowledgements ii
Chinese Abstract iii
English Abstract iv
List of Figures vii
List of Tables viii
List of Abbreviations ix

Chapter 1: Introduction
1.1 PML and the PML-Nuclear Body 1
1.2 SUMOylation drives PML-NB formation 2
1.3 PML TRIM/RBCC domains 4

Chapter 2: Materials and Methods
2.1 Cloning, Expression, and Protein Purification 8
2.2 NMR Spectroscopy 10
2.3 NMR Structure Determination 11
2.4 Analysis of Protein Dynamics by NMR 12
2.5 Residual dipolar couplings (RDC) measurement 13
2.6 Hydrogen-deuterium exchange experiment 14
2.7 Mapping of protein-protein interaction site by NMR chemical shift perturbation (CSP) 15
2.8 Circular Dichroism (CD) measurement 15
2.9 Surface Plasmon Resonance (SPR) assay 16
2.10 Small Angle X-ray Scattering (SAXS) measurement 16

Chapter 3: Results
3.1 PML Domains Architecture and Sequence Alignment 18
3.2 Solution Structure of RING Finger 20
3.3 Dynamics analysis of RING Finger 24
3.4 Concentration dependence of B-box 1 dimerization 25
3.5 Solution Structure of B-box 1 26
3.6 Dynamics analysis of B-box 1 29
3.7 SAXS for PML Domains and Ubc9 32
3.8 Linker between RING Finger and B-box 1 33
3.9 Mapping the interaction sites of PML domains on Ubc9 35
3.10 Mapping the interaction site of Ubc9 on PML domains 37
3.11 SPR quantification of the Ubc9 binding affinity with PML domains 40
3.12 HADDOCK model of the complex 41

Chapter 4: Discussion 43

Chapter 5: Conclusion and Future Plan 50
References 94
Publications

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