实验海洋生物学重点实验室知识库

创伤弧菌（Vibrio vulnificus）是一种嗜温、嗜盐、嗜碱的革兰氏阴性海洋致病菌，广泛分布于河口、海洋，可通过皮肤伤口或肠道感染我国主要的养殖鱼类，致病力强、发病迅速且死亡率高，严重危害水产养殖业的发展。创伤弧菌的强致病性直接与其毒力因子相关，目前研究已发现的毒力因子包括溶细胞素、金属蛋白酶、荚膜多糖等。此外，研究发现创伤弧菌可以通过二型分泌系统分泌一种毒力因子磷脂酶VvPlpA，它以磷脂酰胆碱为特异性底物，在致病性中发挥着重要作用。在感染早期，创伤弧菌通过VvPlpA破坏宿主皮肤或肠道的上皮屏障，入侵组织和血管，催化水解产物还可以作为信号分子引发炎症，进而造成组织坏死和败血症。因此，关于创伤弧菌关键毒力因子VvPlpA的研究，对了解其致病机制和防治策略具有重要意义。

通过比对VvPlpA的氨基酸序列发现，它与弧菌属一类胞外磷脂酶序列的相似度都在60%以上，是弧菌共有的热不稳定溶血素（TLH）家族蛋白，而TLH蛋白的分子结构目前均未得到详细的研究和报道；VvPlpA具有SGNH水解酶家族的典型序列特征，根据该序列特征可以确定VvPlpA的活性位点残基包括丝氨酸（Ser）、甘氨酸（Gly）、天冬酰胺（Asn）以及组氨酸（His），其中Ser和His是主要的催化残基，通常协同位于His序列前的天冬氨酸（Asp）或谷氨酸（Glu），以催化三联体的形式发挥水解功能。VvPlpA蛋白在此Asp/ Glu位点的残基为Gly，显示出非经典三联体模式的序列特征。为深入探究TLH蛋白及非经典模式的催化三联体，更好地了解创伤弧菌致病机制与相应防治手段，本研究以创伤弧菌毒力因子VvPlpA为研究对象，通过结构生物学方法探究并分析其结构和功能的关系。

本研究通过大肠杆菌原核表达系统获得硒代甲硫氨酸的VvPlpA重组蛋白，利用单波长反常散射法获取相位信息，解析了目的蛋白VvPlpA的晶体结构。VvPlpA的N端部分包含多个β折叠结构，具体功能目前未知；C端部分具有典型的α/β/α结构特点，属于SGNH水解酶家族的磷脂酶结构域。通过蛋黄平板和血平板定性实验，我们确定了纯化所得VvPlpA重组蛋白的磷脂酶活性和溶血性。为进一步验证VvPlpA中活性位点残基的关键作用，我们设计构建了对应位点的突变体蛋白，其中Ser152Gly， His392Asn及Asn247Asp表现出极低的催化活性，证明了这些活性位点残基在催化功能中十分必要。

在VvPlpA活性位点His392与上述非典型残基的Gly389附近，我们发现了一个较弱的反常散射信号，推测该位置结合氯离子（Cl^-）。我们通过分子置换法解析了在溴离子条件下纯化和结晶的VvPlpA晶体结构，收集对应溴离子（Br^-）的反常散射信号，验证Br^-对Cl^-的取代，由此确定了Cl^-确实结合在VvPlpA蛋白的活性位点附近。我们利用突变体Gly389Asp模拟具有经典催化三联体的蛋白，测定VvPlpA与Gly389Asp在添加不同Cl^-浓度体系中的磷脂酶活性，结果发现野生型蛋白的催化活性伴随Cl^-浓度的升高而上升，直至达到饱和状态下的最大催化速率，Gly389Asp蛋白活性则不随Cl^-的浓度发生改变。由此说明Cl^-在VvPlpA催化功能中起到关键的作用，同时证明VvPlpA磷脂酶具有新颖的Ser-His-chloride催化三联体模式。

为了解经典催化三联体和二联体在VvPlpA中可能的结构模式，我们解析了突变体Gly389Asp与Gly389Asn的晶体结构，其中氯离子的结合位置均被突变残基的侧链所取代，结构特点与催化活性一致。我们还发现突变体Gly389Asn的活性位点附近结合了一个六乙二醇小分子，显示出狭长的底物结合口袋，为探究催化过程中底物的进入和结合提供了重要的结构基础。

Vibrio vulnificus is a mesophilic, halophilic, basophilic gram-negative marine pathogen that is widely distributed in estuaries and oceans. V. vulnificus can infect major economic fish through skin wounds or intestinal tracts. The infection is characterized by rapid onset and high mortality, and seriously endangers the development of aquaculture. The strong pathogenicity of V. vulnificus is directly related to its multiple virulence factors. The V. vulnificus phospholipase (VvPlpA) plays an important role in its pathogenicity. VvPlpA is secreted by the type II secretion system and catalyzes phosphatidylcholine as a specific substrate. The hydrolysate acts as a signaling molecule to stimulate the occurrence of inflammation, which in turn causes intense infection such as tissue necrosis and sepsis. Study on the virulence factor is conducive to understanding the pathogenic mechanisms of V. vulnificus.

According to amino acid sequence alignment, VvPlpA has over 60% sequence similarity with homologs in vibrio, and belongs to the thermolabile hemolysin (TLH) family. But no structural information for this class of virulence factors is available. Sequence analysis showed VvPlpA has a typical sequence characteristic of the SGNH hydrolase superfamily. This superfamily is characterized by containing four conserved catalytic residues, serine, glycine, asparagine and histidine. Serine and histidine are the key catalytic residues, and usually cooperate with aspartate or glutamate preceding the histidine, thus forming a Ser-His-Asp/Glu catalytic triad. However, the aspartate/glutamate is substituted by a non-classical glycine in VvPlpA, and exhibits a non-canonical active site pattern.

In this study, the recombinant protein VvPlpA was expressed in Escherichia coli. The crystal structure was solved at a high resolution by molecular replacement. The structure shows that the N-terminal domain mainly contains multiple β-sheets with unknown function, while the C-terminal domain has typical α/β/α structure characteristics and belongs to the SGNH hydrolase superfamily. The Ser152Gly, His392Asn and Asn247Asp mutants further verify the key role of the conserved residues in catalysis.

Near the conserved His392 and atypical Gly389 of VvPlpA, we found a weak anomalous scattering signal and presumed it to be a chloride ion. Then the solved structure of bromine-substituted VvPlpA showed a strong anomalous scattering signal of bromine at the same position, indicating the substitution of bromine ion for chloride ion. The importance of Cl^- was tested by monitoring the chloride dependence of enzyme activity. The results showed that the activity of the wild type protein increases with increasing chloride ion concentration, while the activity of the Gly389Asp protein does not. It indicated that the chloride ion plays a key role in the catalytic function and forms an unusual Ser-His-chloride catalytic triad.

At the same time, we analyzed the crystal structures of the Gly389Asp and Gly389Asn mutants which were used to simulate classical catalytic triad and dyad. We found that the chloride ion at the binding site was replaced by the side chain of each mutated residue. The structural characteristics were consistent with the performance of catalytic activity. A hexaethylene glycol was bound to the narrow pocket near the active site of the Gly389Asn mutant, providing information on substrate binding mode.

第1章 绪论.................................................................................. 1

1.1 创伤弧菌............................................................................................... 1

1.1.1 创伤弧菌概述................................................................................ 1

1.1.2 创伤弧菌的危害和防治................................................................ 1

1.1.3 毒力因子与致病机制.................................................................... 2

1.2 磷脂酶毒力因子................................................................................... 3

1.2.1 细菌磷脂酶及作用机制................................................................ 3

1.2.2 弧菌的热不稳定溶血素................................................................ 5

1.2.3 创伤弧菌磷脂酶VvPlpA.............................................................. 6

1.3 SGNH水解酶超家族............................................................................ 7

1.3.1 SGNH水解酶概述......................................................................... 7

1.3.2 SGNH水解酶催化活性位点......................................................... 8

1.4 活性位点的催化三联体模式............................................................... 8

1.4.1 经典型催化三联体........................................................................ 8

1.4.2 非经典型催化三联体.................................................................... 8

1.5 本研究目的和意义............................................................................. 10

第2章 材料与方法.................................................................... 11

2.1 实验材料............................................................................................. 11

2.1.1 实验菌株与载体.......................................................................... 11

2.1.2 实验仪器...................................................................................... 12

2.1.3 试剂及耗材.................................................................................. 13

2.2 实验方法............................................................................................. 15

2.2.1 重组质粒载体的构建.................................................................. 15

2.2.2 重组蛋白的表达.......................................................................... 20

2.2.3 目的蛋白的纯化.......................................................................... 23

2.2.4 目的蛋白的结晶.......................................................................... 25

2.2.5 衍射数据的收集与处理.............................................................. 27

2.2.6 结构解析与修正.......................................................................... 29

2.2.7 蛋白结构分析.............................................................................. 30

2.2.8 磷脂酶VvPlpA催化活性的测定............................................... 30

第3章 VvPlpA蛋白结构与功能的研究结果和讨论.............. 35

3.1 创伤弧菌VvPlpA的生物信息学分析.............................................. 35

3.1.1 VvPlpA序列信息......................................................................... 35

3.1.2 理化性质分析.............................................................................. 36

3.1.3 二级结构预测分析...................................................................... 37

3.1.4 结构域分析.................................................................................. 38

3.2 VvPlpA蛋白的表达、纯化与活性鉴定............................................ 38

3.2.1蛋白表达与纯化........................................................................... 38

3.2.2目的蛋白的生理活性鉴定........................................................... 40

3.3 蛋白结晶条件的初筛及优化............................................................. 40

3.3.1 VvPlpA和Se-Met-VvPlpA的结晶初筛和优化......................... 40

3.3.2 突变体Gly389Asp、Gly389Asn的结晶初筛和优化............... 43

3.3.3 溴代型蛋白VvPlpA-Br^-的结晶初筛和优化.............................. 45

3.4 晶体结构解析..................................................................................... 47

3.4.1 晶体衍射数据收集与处理.......................................................... 47

3.4.2 结构相位确定与修正.................................................................. 49

3.4.3 结构模型的质量评估.................................................................. 49

3.5 VvPlpA晶体结构与功能分析............................................................ 51

3.5.1 VvPlpA整体结构......................................................................... 51

3.5.2 VvPlpA活性位点结合氯离子的验证......................................... 53

3.5.3 Ser-His-chloride型催化三联体................................................... 57

3.5.4 VvP1pA底物结合区域的结构信息............................................ 62

3.5.5 VvP1pA蛋白的同源结构比对分析............................................ 64

第4章 结论与展望.................................................................... 67

4.1 结论..................................................................................................... 67

4.2 创新点................................................................................................. 68

4.3 展望..................................................................................................... 68

参考文献...................................................................................... 71

致 谢............................................................................................ 77

作者简历及攻读学位期间发表的学术论文与研究成果.................. 79