臺灣博碩士論文加值系統

Uracil-DNA glycosylase (UDG) 為一種保守DNA修復蛋白，對於皰疹病毒 DNA
之複製非常重要; 因此，它被認為是一個良好抗病毒標的。在先前的研究中，SAUGI（Staphylococcus aureus uracil-DNA glycosylase inhibitor） 已被發現為一具有抑制四個不同物種之 UDG 活性的 DNA 擬態蛋白質，並且對於兩種人類皰疹病毒（人類皰疹病毒第四型； EBV 和單純皰疹病毒； HSV ） 之 UDG 活性，表現出很好的抑制效果。本篇論文中我們發現 SAUGI 對於三種人類皰疹病毒之 UDG 表現出截然不同的結合強度和抑制能力。特別的是，雖然 γ 皰疹病毒 UDG（ EBVUDG 和 KSHVUDG ） 胺基酸序列相似度高達 60%，但 SAUGI 對於此兩種 UDG 的結合能力和抑制效果有很大差異。我們進而利用 X-ray crystallography 方式解析 SAUGI/EBVUDG NΔ24 和 SAUGI/KSHVUDG NΔ18 之複合體結構。結構分析中顯示，與 SAUGI/EBVUDG NΔ24 複合體相比， SAUGI 與 KSHVUDG 之亮胺酸環延伸區域 （leucine loop extension）中參與 KSHVUDG 活性之部分重要胺基酸沒有交互作用; 此外，我們也發現 KSHVUDG 與SAUGI β sheet 1之結合區域 （binding interface） 相較於 EBVUDG帶有較多之負電荷。其可以解釋為何 SAUGI 對於 KSHVUDG 表現出較低之結合和抑制能力。而在 SAUGI/EBVUDG NΔ24 複合體結構中， 相較於其他 SAUGI/UDG 複合體之結構，我們也發現 SAUGI 與 EBVUDG 之亮胺酸環 （leucine loop） 上胺基酸有額外之交互作用，其可以解釋為何 SAUGI 對 EBVUDG 有較好之結合親和力（binding affinity）。藉由這些結構資訊，我們進一步利用蛋白質工程調整 SAUGI 活性，使其對人類 UDG 無抑制效果但仍同時保有對 EBVUDG 活性之抑制能力。在這篇研究中，我們的結果不僅以分子基礎解析 SAUGI 為何對於兩種皰疹病毒 UDG 具有不同之交互作用，同時也為開發可用作生物醫學應用之人類皰疹病毒 UDG 抑制劑衍生物提供了框架。

Uracil-DNA glycosylases (UDGs) are conserved DNA-repair proteins that play critical roles in viral DNA efficient replication and epigenetic maintenance in herpesviruses. They have been implicated as good antiviral targets. Previously, Staphylococcus aureus SAUGI was identified as a DNA mimic protein that targets UDG from four different species, and that represents the greatest inhibitory activity on the UDGs from two human herpesviruses (Epstein–Barr virus; EBV and Herpes simplex virus; HSV). In this study, we show that SAUGI displays strikingly differential effects on two γ-herpesvirus UDGs (EBVUDG and KSHVUDG) catalytic activity. We further performed two complex structures of SAUGI/EBVUDG NΔ24 and SAUGI/KSHVUDG NΔ18. Analysis of the structures reveals that SAUGI only partially interacts with the critical residues that are responsible for the catalytic activity of KSHVUDG as compared to SAUGI/EBVUDG NΔ24 complex. In addition, we found that the leucine loop extension of EBVUDG provides an additional contact region with SAUGI not seen in other SAUGI/UDG complexes could explain the strong binding capacity of SAUGI to EBVUDG. Furthermore, we also modulate the inhibitory effect of SAUGI on human UDG and EBVUDG based on this structural information. Thus, our results not only provide the molecular basis showing that the differential interactions of SAUGI on two γ-herpesvirus UDGs but also a framework for the development of derivatives that could serve as human herpesvirus UDGs inhibitors for biomedical applications.

Verification letter from oral examination committee
List of Tables VI
List of Figures VII
Abbreviations X
Chinese abstract 1
Abstract 3

Chapter 1 Introduction 5
1.1 DNA mimic protein 5
1.2 Recent studies of DNA mimic protein S. aureus SAUGI 6
1.3 Herpesvirus UDGs and their importance in viral replication 7
1.4 The potential application of SAUGI in anti EBV-related diseases 9
1.5 Specific aim 10

Chapter 2 Experimental Procedures 12
2.1 SAUGI and the UDGs preparation and expression 12
2.2 Gel filtration analysis 13
2.3 Inhibitory effect analysis 13
2.4 His-pulldown assay 15
2.5 Microscale thermophoresis (MST) analysis 15
2.6 Crystallization and structure determination 17

Chapter 3 Results and discussion 19
3.1 Investigation of the interactions between SAUGI and herpesvirus UDGs 19
3.2 The differential inhibition effects of SAUGI on human herpesvirus UDGs 20
3.3 Differential binding affinity between SAUGI and herpesvirus UDGs 21
3.4 Overall crystal structures 22
3.5 The binding interface between SAUGI and EBV and KSHV UDGs 24
3.6 Structural difference between SAUGI in complex with EBVUDG NΔ24 and KSHVUDG NΔ18 26
3.7 Other differential interactions between SAUGI/EBVUDG NΔ24 and SAUGI/KSHVUDG NΔ18 complexes 28
3.8 Protein engineering to modify the differential inhibition effects of SAUGI on EBV and human UDGs 29
3.9 In silico approach and inhibitory effect assay of SAUGI Q52K to EBVUDG and human UDG 30

Chapter 4 Conclusion 32

References 34
Tables 44
Table 1. The 22 previously known DNA mimic proteins 45
Table 2. Binding affinity of SAUGI to SAUDG, human UDG, HSVUDG and EBVUDG determined by BIACORE 47
Table 3. Human herpesviruses and associated disease 48
Table 4. MST analysis of the binding affinity between SAUGI and three herpesvirus UDGs 49
Table 5. X-ray data collection and refinement statistics for SAUGI/KSHVUDG NΔ18 and SAUGI/EBVUDG NΔ24 complexes 50
Table 6. The hydrogen bonding and van der Waals (VDW) interactions between SAUGI and conserved EBVUDG motifs 51
Table 7. The hydrogen bonding and van der Waals (VDW) interactions between SAUGI and conserved KSHVUDG motifs 52
Table 8. Design of interface mutants on SAUGI 54

Figures 55
Figure 1. An example of a DNA mimic protein blocks its target DNA binding protein from DNA by direct competition 56
Figure 2. Multiple sequence alignment of HSVUDG, Human UDG, HCMVUDG, EBVUDG and KSHVUDG 57
Figure 3. Purification of SAUGI/EBVUDG NΔ24 and SAUGI/KSHVUDG NΔ18 protein complexes 58
Figure 4. Photography of SAUGI/EBVUDG NΔ24 and SAUGI/KSHVUDG NΔ18 crystals and X-ray diffraction pattern 60
Figure 5. Characterizes the interactions between SAUGI and three human herpesvirus UDGs by His pull-down assay 62
Figure 6. The gel filtration analysis demonstrating the interactions of SAUGI to HSV, EBV, HCMV and KSHV UDGs 64
Figure 7. The inhibitory effect analysis of SAUGI on EBVUDG NΔ24 66
Figure 8. The inhibitory effect analysis of SAUGI on KSHVUDG NΔ18 67
Figure 9. The inhibitory effect analysis of SAUGI on HCMVUDG 68
Figure 10. Sequence and structural comparison of herpesvirus and human UDGs 69
Figure 11. Determination of the binding constants between SAUGI and three human herpesvirus UDGs using microscale thermophoresis (MST) 72
Figure 12. Crystal complex structures of SAUGI/KSHVUDG NΔ18 and SAUGI/EBVUDG NΔ24 73
Figure 13. Structural comparison of complex structures of SAUGI/EBVUDG NΔ24, SAUGI/Human UDG, SAUGI/HSVUDG and SAUGI/KSHVUDG NΔ18 75
Figure 14. Comparison of shape and charge distribution of SAUGI in complex with KSHVUDG NΔ18 and EBVUDG NΔ24 complexes 77
Figure 15. Structural details of the interaction between SAUGI β sheet 1 and both UDGs 79
Figure 16. Structure comparison of the leucine loop in SAUGI/EBVUDG NΔ24 and SAUGI/KSHVUDG NΔ18 80
Figure 17. Sequence alignment and structure comparison of leucine loop in SAUGI/EBVUDG NΔ24 and SAUGI/KSHVUDG NΔ18 81
Figure 18. SAUGI targets the Gln212 residue of EBVUDG, but not the Gln209 of KSHVUDG 83
Figure 19. Structure details of leucine loop extension in SAUGI/EBVUDG NΔ24 and SAUGI/KSHVUDG NΔ18 84
Figure 20. Other differential interactions between SAUGI and the leucine loop extensions on EBVUDG and KSHVUDG 85
Figure 21. Differential interactions between SAUGI and the leucine loop extensions on EBVUDG and KSHVUDG 87
Figure 22. The conserved interaction regions between SAUGI/EBVUDG NΔ24 and SAUGI/Human UDG 88
Figure 23. Comparison of the SAUGI/SAUDG (PDB entry 3WDG), SAUGI/Human UDG (PDB entry 5AYR), SAUGI/HSVUDG (PDB entry 5AYS) and SAUGI/EBVUDG NΔ24 structures 89
Figure 24. Differential interaction region between SAUGI/EBVUDG NΔ24 and SAUGI/Human UDG 90
Figure 25. Investigation of the inhibitory effects of SAUGI Q52K on human UDG and EBVUDG by UDG activity assay 91

Appendices 92

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