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研究生(外文):Der-Shyang Kao
論文名稱:Spartan UBZ4 類型 UBD 與泛素交互作用的結構探討
論文名稱(外文):Structural basis for the interaction of UBZ4-type UBD from Spartan with ubiquitin
指導教授(外文):Chun-Hua Hsu
口試委員(外文):Shang-Te Danny HsuShih-Che Sue
中文關鍵詞:泛素鋅指UBZ 區塊Spartan 調控蛋白核磁共振等溫滴定熱卡計
外文關鍵詞:ubiquitinzinc fingerUBZ domainSpartanNMRITC
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鋅指 (Zinc finger) 是一類重要的區塊,除了有些鋅指具DNA辨識功能之外,也有些被發現參與在各種不同的蛋白質交互作用。最近在DNA損傷反應中,一個具有以鋅指區塊結合泛素的調控蛋白Spartan被發現,而其泛素結合之鋅指區塊(UBZ)被歸類為一新穎UBZ4類型泛素結合區塊 (UBD)。然而,目前對於UBZ4蛋白質家族與泛素分子辨識,瞭解的結構細節仍有限。本研究希望以跨領域的方法,探討人類Spartan蛋白的UBZ與泛素的交互作用。In vitro pull down試驗的結果指出,Spartan UBZ與泛素確實會互相結合。而利用等溫滴定熱卡計 (ITC)實驗測得解離常數 (Kd) 為6.349 μM以及結合化學計量數為1.782,顯示Spartan UBZ可能具有兩個泛素結合位。此外,我們收集了Spartan UBZ的多維核磁共振圖譜後,已完成蛋白質之化學位移判讀。以此為基礎進一步利用化學位移擾動實驗,我們定位出Spartan UBZ與泛素於結構上各自的結合區域。將位於α螺旋並觀察到顯著化學位移擾動的L476進行數種胺基酸的定點突變,再利用ITC進行量測,可觀察到其結合親和性的下降或破壞。此外,UBZ的D473與泛素的R42、R72也有化學位移的擾動變化,暗示靜電吸引力可能也對兩個蛋白質的結合做出貢獻,而ITC量測也觀察到D473的定點突變顯著破壞Spartan UBZ與泛素的結合。綜合以上結果並以這些訊息建構分子對接模型 (HADDOCK model),顯示Spartan UBZ的α螺旋藉由疏水作用力與靜電吸引力與泛素結合。我們的研究除了能提供Spartan UBZ區塊與泛素分子辨識的結構資訊,也擴展了我們對於UBZ4類型鋅指蛋白質家族的瞭解。

Zinc fingers are important protein domains, which not only recognize DNA sequences, but also mediate various protein–protein interactions. Recently, an UBZ (ubiquitin-binding zinc finger) domain as a new type of ubiquitin-binding domain (UBD) of a novel regulator protein, Spartan, was identified in DNA damage responses, which belongs to the UBZ4 protein family. However, the structural details of molecular recognition for UBZ4 protein family with ubiquitin are still limited. Here, we aim to study the interaction of Spartan UBZ domain and ubiquitin by multidisciplinary approaches. The data from in vitro pull down assay indicated the interaction of Spartan UBZ domain and ubiquitin. Isothermal titration calorimetry (ITC) measurements revealed that Spartan UBZ domain binds to ubiquitin with a dissociation constant (Kd) of 6.349 μM and stoichiometry (n) of 1.782, suggesting two ubiquitin binding site on Spartan UBZ. Using nuclear magnetic resonance (NMR), the resonance assignment of Spartan UBZ domain was completed. Based on the further chemical-shift perturbation experiments, binding interfaces for Spartan UBZ domain and ubiquitin were mapped into protein structures. Mutations of significantly perturbed hydrophobic L476 on the Spartan UBZ α-helix decrease or spoil the binding affinity monitored by ITC measurements. In addition, important chemical-shift perturbations on D473 of Spartan UBZ domain and R42, K48 of ubiquitin are also observed. This result suggests that the electrostatic attraction may be contributed to protein interaction, and ITC measurements also show D473 mutageneses significantly abolish the binding between Spartan UBZ and ubiquitin. Taking together, the information-driven HADDOCK model shows the binding mode as that Spartan UBZ domain recognizes ubiquitin through hydrophobic and electrostatic interaction by the α-helix. Our studies shed insight into the recognition of Spartan UBZ domain with ubiquitin and extend our understanding of the UBZ4-type zinc finger protein family.

誌謝 i
Abstract ii
中文摘要 v
Table of contents vi
List of figure ix
Introduction 1
Zinc finger (ZnF) is one of the most abundant ubiquitin binding domains (UBDs) 1
Ubiquitin-binding ZnF (UBZ) domain regulates DNA damage responses 2
Spartan is a novel TLS regulator containing an UBZ4 domain 4
Purpose of this study 6
Material and methods 7
Sequence alignment 7
Molecular cloning 7
Spartan UBZ protein overexpression 7
Ni-NTA affinity column purification for His-tag fusion proteins 8
Thrombin digestion 9
Protein concentration determination 9
Atomic absorption spectroscopy (AAS) 9
Circular dichroism (CD) spectroscopy 9
In vitro pull down assay 10
Isotope labeling of protein NMR samples 11
Sequential protein backbone assignments 13
Protein secondary structure prediction by web server TALOS+ 13
Alpha proton chemical shift index (Hα CSI) value calculation 13
NMR titration experiments 14
Isothermal titration calorimetry (ITC) 15
Molecular docking 15
Results 17
Overexpression and purification of Spartan UBZ and ubiquitin 17
Spartan UBZ domain requires zinc ion to stabilized its secondary structures 18
Spartan UBZ physically interacts with ubiquitin in vitro 18
Overexpression and purification isotope-labeled Spartan UBZ and ubiquitin 19
Protein NMR backbone resonance assignments 19
Protein secondary structure prediction by NMR chemical shift data 20
NMR titration and chemical shift perturbation (CSP) analysis 21
ITC measurements show binding parameters and effect of mutagenesis 22
HADDOCK model of Spartan UBZ-ubiquitin complex indicates electrostatic and hydrophobic attraction may significantly contribute the interaction 24
Discussion 27
Interaction of Spartan UBZ and ubiquitin may act in two steps 27
Spartan UBZ may bind to mono- and poly-ubiquitin 28
The binding of ubiquitin may induce more α helical structure of Spartan UBZ 29
Figures 30
References 65
Appendixes 73

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