中国科学院海洋研究所机构知识库

白斑综合征病毒WSSV（white spot syndrome virus）是全世界范围内甲壳类动物的重要病原，至今未获得对其有效的防治措施，因此开发抗WSSV药物对于甲壳类动物养殖产业的健康可持续发展具有重要意义。WSSV基因组能够编码胸苷酸合成通路中的关键酶dUTP焦磷酸酶（dUTP pyrophosphatase，dUTPase），该酶存在于绝大多数生物中，催化dUTP水解产生dUMP和焦磷酸PPi，对于DNA的合成与稳定起着不可或缺的作用，是抗病原体药物开发的重要靶标。开发以dUTPase为靶标的抗WSSV药物，需要深入了解WSSV和宿主dUTPase的结构特点。

本文通过解析WSSV和凡纳滨对虾Litopenaeus vannamei（WSSV侵染的主要养殖对象）dUTPase保守结构域的晶体结构，结合氨基酸定点突变和酶动力学分析等方法，深入探究两者的结构与功能特点，阐明其结构差异，不但从分子水平丰富了对dUTPase家族分类和催化反应机制的认识和理解，并且为抗WSSV药物的开发提供了准确的蛋白结构信息。

氨基酸序列比对显示，WSSV基因组中wsv112基因编码的蛋白序列N端171个氨基酸（wDUT）构成的dUTPase属于三聚体家族，包含酶催化活性必需的5个基序（I-V）。wDUT在基序V前具有特异性插入序列pre-V，该序列由20个氨基酸（Tyr134-Asn153）组成。wDUT晶体结构分析发现，pre-V形成独特的β发夹结构，引导其后C端序列产生了新颖的走向，导致基序V覆盖到自身亚基参与形成的底物结合口袋上，从而使wDUT活性位点仅由两个亚基组成，这与三聚体dUTPase活性位点由三个亚基组成的传统认识截然不同。该结果表明wDUT是同源三聚体dUTPase家族中新的亚型，扩充了对dUTPase家族分类的认识。

通过结构比对和氨基酸定点突变体的酶动力学分析发现，wDUT活性位点的氨基酸组成和催化反应机制与传统型三聚体dUTPase基本一致。然而，pre-V截短后，酶催化效率及蛋白热稳定性会显著下降，这表明pre-V截短体中由三个亚基形成的活性位点并不具有高水平酶催化效率，暗示wDUT发生了适应于新型活性位点组织方式的氨基酸替代。进一步利用wDUT和大肠杆菌dUTPase（传统型三聚体代表）突变体进行酶动力学分析发现，传统型三聚体dUTPase基序IV中保守Arg/Lys的功能被wDUT基序I前特异的Arg24替代，并且该wDUT活性位点周围氨基酸的重排与其新颖的C端走向相适应，共同维持了wDUT高水平的酶催化效率，该结果表明wDUT发生了序列插入与氨基酸突变的协同进化。

不同于WSSV的wDUT蛋白，宿主凡纳滨对虾dUTPase序列C端的146个氨基酸（LvDUT）为传统的同源三聚体dUTPase保守结构域。通过LvDUT多种反应状态晶体结构的比较，我们首次发现底物dUTP的结合能够引起dUTPase蛋白主体部分产生两种新型的构象变化：一是活性位点区域Ser132-His138由无规则卷曲变为α螺旋，二是三聚体中央通道一端发生“开合”。这两种构象变化共同促进了dUTP水解反应的发生。

wDUT和LvDUT结构比对表明，两者在传统的dUTPase抑制剂设计位点，即底物结合口袋和三聚体中央通道上差异较小，不利于针对wDUT抑制剂的开发和使用。但是，在wDUT独特的C端走向上，Lys150-Asn155与蛋白主体形成的空间中存在合适的小分子化合物结合位点，可以作为wDUT潜在的特异性抑制剂结合靶位，这为抗WSSV特异性药物的开发奠定了分子结构基础。

WSSV (white spot syndrome virus) is an important pathogen for crustaceans all over the world and has not been effectively controlled so far. The development of antiviral drugs is of great significance for the healthy and sustainable development of the crustacean culture industry. WSSV genome encodes dUTPase (dUTP Pyrophosphatase), a key enzyme in the thymidylate biosynthesis pathway. dUTPases that exist in most organisms, can catalyze the hydrolysis of dUTP to dUMP and pyrophosphate PPi, playing essential roles for DNA synthesis and stability. It is an important target for the development of anti-pathogenic drugs.The development of anti-WSSV drugs targeting to its dUTPase requires detailed understanding of the structure characteristics of WSSV and host dUTPases.

In the present study, structural and functional characteristics of the conserved dUTPase domains from WSSV and Litopenaeus vannamei, an important culture species infected by WSSV, were studied by crystal structure analysis, residue site-directed mutagenesis, enzyme kinetic analysis and other methods. The results enriched the current knowledge of the classification and catalytic reaction mechanism of the dUTPase family and provided the accurate protein structural basis for the development of anti-WSSV drugs.

The amino acid sequence alignment showed that, the N-terminal 171 residues of the wsv112-encoded protein (wDUT) in the WSSV belongs to the trimeric dUTPase family with five conserved motifs (I-V) which are necessary for the enzyme catalysis. Interstingly, wDUT harbors a unusual 20-residue (Tyr134-Asn153) insert (pre-V) at the position preceding the C-terminal catalytic motif V. wDUT crystal structure analysis revealed that the insert pre-V forms an unusual β-hairpin structure in the domain-swapping region and thereby facilitates a unique orientation of the adjacent C-terminal segment, bringing the catalytic motif V onto the active site of its own subunit. As a result, wDUT forms a new subclass of the homotrimeric dUTPase family with the noncanonical two-subunit active site assembly, distinct from the classic three-subunit active site assembly. This finding expands the current classification knowledge of the dUTPase family.

The residues at the active site and the catalytic mechanism of wDUT are basically the same as the classic trimeric dUTPase through structure comparison and enzyme kinetic analysis of site-directed mutants. However, deletion of the pre-V insert significantly reduces wDUT’s enzymatic activity and thermal stability. This result suggested that the classic three-subunit active site in wDUT without the pre-V insert lost the high catalytic power, implying that wDUT has residue substitutions adapting to its novel two-subunit active site. Through the enzymatic kinetic analysis of wDUT and Escherichia coli dUTPase (prototype model in trimeric family) mutants, a conserved arginine/lysine located in motif IV of the classic dUTPases was proved to be functionally substituted by an unusual arginine (residue Arg24) at the position preceding motif I in wDUT. This rearrangement of wDUT residues occurs in the second shell of the active site is compatible with its novel C-terminal orientation to maintains the high catalytic power, suggesting that the evolution of wDUT should be considered the combined result of sequence insertion and amino acid substitution.

The amino acid sequence alignment and crystal structure comparison showed that, the C-terminal 146 residues of dUTPase from L. vannamei (LvDUT) constitute a traditional trimeric dUTPase domain, distinct from the wDUT of WSSV. The comparison of LvDUT crystal structures in various reaction stages indicated that, dUTP binding can induce two kinds of novel conformational changes in the main body of dUTPases: the loop-to-alpha helix conformational change of the Ser132-His138 in the active site region and the "open-close" change of the trimer central channel. These conformational changes together promoted the reaction of dUTP hydrolysis.

The structure comparison between wDUT and LvDUT showed that, both share essentially identical structures in two traditional sites of trimeric dUTPases for inhibitor design, including the substrate binding pocket and the trimer central channel, making it difficult to develop and use specific inhibitors targeting to wDUT. However, an appropriate small-molecule binding site is found in the space between the Lys150-Asn155 region and the mainbody in wDUT and can serve as a potential antiviral target for the wDUT inhibitor design. This finding provides the structural basis for developing specific antiviral drugs against WSSV infections.

摘  要... I

ABSTRACT.. III

第一章  绪论... 1

1.1  白斑综合征病毒WSSV的研究现状... 1

1.1.1  WSSV的致病机制... 1

1.1.2  WSSV的基因组特征... 4

1.1.3  WSSV的蛋白质组特征... 5

1.1.4  WSSV的防治... 9

1.2  dUTP焦磷酸酶（dUTPase）的研究概述... 11

1.2.1  dUTPase的作用与分类... 11

1.2.2  dUTPase的催化机制... 13

1.2.3  dUTPase是重要的药物靶标... 15

1.2.4  dUTPase具有酶催化活性以外的其他功能... 16

1.3  本课题的研究目的和意义... 18

第二章  材料与方法... 21

2.1  实验材料... 21

2.1.1  模板和菌株... 21

2.1.2  质粒和引物... 21

2.1.3  试剂耗材... 21

2.1.4  仪器设备... 22

2.2  实验方法... 24

2.2.1  目的基因克隆... 24

2.2.2  重组表达载体构建... 26

2.2.3  定点突变表达载体构建... 28

2.2.4  重组蛋白体外表达与纯化... 29

2.2.5  dUTPase酶催化反应检测... 32

2.2.6  dUTPase酶动力学参数测定... 34

2.2.7  重组蛋白热稳定性检测... 35

2.2.8  蛋白晶体制备... 37

2.2.9  晶体衍射数据收集与处理... 40

2.2.10  蛋白晶体结构解析... 42

第三章  WSSV dUTPase结构与功能研究的结果及分析... 45

3.1  WSSV dUTPase序列分析与蛋白纯化... 45

3.1.1  WSSV dUTPase序列信息与分析... 45

3.1.2  WSSV dUTPase保守区（wDUT）重组蛋白的表达与纯化... 50

3.1.3  大肠杆菌dUTPase（eDUT）重组蛋白的表达与纯化... 51

3.1.4  重组蛋白wDUT的分子聚集状态分析... 51

3.1.5  重组蛋白wDUT和eDUT的催化活性确认和酶动力学分析... 52

3.2  无底物结合（apo）wDUT的晶体结构解析... 54

3.2.1  apo wDUT蛋白晶体的制备... 54

3.2.2  apo wDUT晶体衍射数据的收集与处理... 54

3.2.3  分子置换法解析apo wDUT晶体结构... 56

3.2.4  apo wDUT晶体结构的质量评价... 57

3.3  apo wDUT晶体结构分析... 59

3.3.1  apo wDUT的整体结构... 59

3.3.2  wDUT插入序列pre-V形成独特的结构... 61

3.3.3  基于apo wDUT结构的C端走向预测... 62

3.4  wDUT复合物晶体的结构解析... 63

3.4.1  wDUT复合物晶体制备... 63

3.4.2  wDUT复合物晶体衍射数据的收集与处理... 64

3.4.3  分子置换法解析wDUT复合物的晶体结构... 67

3.4.4  wDUT复合物晶体结构中配体的确认... 68

3.4.5  wDUT复合物晶体结构的质量分析... 69

3.5  wDUT复合物的结构与功能分析... 71

3.5.1  wDUT新颖C端走向产生的新型活性位点组织方式... 71

3.5.2  wDUT活性位点的氨基酸组成... 77

3.5.3  wDUT的催化反应机制... 79

3.5.4  wDUT插入序列pre-V的作用分析... 81

3.5.5  wDUT适应于新颖C端走向的氨基酸重排分析... 82

3.6  本章小结... 87

第四章 凡纳滨对虾dUTPase结构与功能研究的结果及分析... 89

4.1  凡纳滨对虾dUTPase序列分析与蛋白纯化... 89

4.1.1  凡纳滨对虾dUTPase序列分析... 89

4.1.2  凡纳滨对虾dUTPase保守区（LvDUT）重组蛋白的表达与纯化... 93

4.1.3  重组蛋白LvDUT分子聚集状态分析... 94

4.1.4  重组蛋白LvDUT的酶催化活性确认和酶动力学参数测定... 95

4.2  LvDUT晶体结构解析... 95

4.2.1  LvDUT晶体的制备... 95

4.2.2  LvDUT晶体衍射数据的收集与处理... 96

4.2.3  分子置换法解析LvDUT晶体结构... 99

4.2.4  LvDUT晶体结构的质量评价... 100

4.3  LvDUT结构与功能分析... 102

4.3.1  LvDUT整体结构的构象变化... 102

4.3.2  LvDUT活性位点的构象变化... 104

4.3.3  LvDUT具有真核生物三聚体dUTPase的保守特征... 106

4.3.4  LvDUT与wDUT的结构比较... 107

4.4  本章小结... 109

第五章  讨论... 111

5.1  WSSV和凡纳滨对虾dUTPase均具有额外基序但作用不同... 111

5.1.1  wDUT形成了三聚体家族中新的亚型... 111

5.1.2  wDUT具有发挥酶催化功能以外其他作用的结构基础... 112

5.1.3  WSSV和凡纳滨对虾dUTPase中存在未知功能的额外基序... 113

5.2  wDUT具有序列插入和氨基酸突变的协同进化... 114

5.3  LvDUT产生由底物dUTP结合引起的新型构象变化... 115

5.4  wDUT具有潜在的特异性抑制剂结合靶位... 116

第六章  总结及展望... 119

6.1  结论... 119

6.2  创新点... 119

6.3  展望... 120

参考文献... 121

致  谢... 135

作者简历及攻读学位期间发表的学术论文与研究成果    137