实验海洋生物学重点实验室知识库

  创伤弧菌（Vibrio vulnificus）是一类宿主广泛且致病力极强的重要海洋病原微生物，可以通过食入和伤口接触感染人类以及多种海洋经济鱼类，感染后迅速发病且致死率极高，对公共健康和水产养殖产业的发展造成极大的威胁。因此，深入了解创伤弧菌的致病机制和开发特异性抗菌药物成为亟待解决的重要问题。

  荚膜是创伤弧菌表面的特殊结构，也是造成创伤弧菌强致病性的重要毒力因子，但合成荚膜的相关蛋白的结构和功能研究还相对较少。创伤弧菌Wzb、Wzc（VvWzb、VvWzc）是一对酪氨酸磷酸酶/激酶组合，VvWzc可以通过自磷酸化其C端富含酪氨酸的尾部（Y-cluster）使其呈现高磷酸化状态，VvWzb可以去磷酸化VvWzc使其呈现低磷酸化状态，VvWzc在高低磷酸化状态的循环中调节荚膜多糖的产生，但其机制尚不明确。对VvWzb、VvWzc进行深入的结构与功能研究将为创伤弧菌的荚膜多糖合成提供重要线索，增强对创伤弧菌致病性的了解，并为特异性抗菌药物的开发提供有效的靶标信息。

      VvWzb属于低分子量酪氨酸磷酸酶（LMWPTP）家族，含有该家族保守的催化特征序列（P-loop、D-loop），但在底物识别相关的关键区域W-loop上存在额外的氨基酸序列（⁴⁰EKSR⁴³）插入。进一步结构分析发现，⁴⁰EKSR⁴³插入区在底物结合后发生了明显的构象变化，重塑了蛋白活性口袋的形状和表面电荷分布。这是LMWPTP家族中首次发现底物可以诱导W-loop区域的显著结构变化。酶动力学分析发现，⁴⁰EKSR⁴³插入区与VvWzb蛋白活性直接相关，但其对活性的影响并不是通过序列特异性导致的，推测是以整体的空间效应提高催化能力。Escherichia coli Wzb（EcWzb）W-loop较VvWzb W-loop缺少⁴⁰EKSR⁴³序列，将该序列插入EcWzb W-loop相应位置，发现EcWzb催化活性明显上升，表明VvWzb W-loop ⁴⁰EKSR⁴³序列可以作为通用的“酶活提高元件”应用于该家族其他蛋白的改造。进一步的系统发育分析显示，LMWPTP家族以W-loop类型为标记分为三种类型，VvWzb以独特的W-loop类型在LMWPTP家族中占据独立的分类地位，代表LMWPTP家族的新类型。

      VvWzc属于细菌酪氨酸激酶（BY-kinase）家族，含有该家族保守的周质结构域、跨膜结构域和激酶结构域。激酶结构域（VvWzc_KD）是该蛋白通过磷酸化状态变化实现功能的重要区域，因而本课题主要针对该结构域进行研究。VvWzc_KD在含Y-cluster时呈现C₈对称的环状八聚体结构，每个单体结合有一个ADP分子，模拟蛋白发挥激酶功能的工作状态，其Y-cluster表现出部分磷酸化状态并插入蛋白分子的活性中心处，这与E. coli Wzc_KD^K540M非磷酸化八聚体的Y-cluster结构一致，直观展现出部分磷酸化的VvWzc_KD仍能形成八聚体的结构基础。这是在BY-kinase家族中首次观察到由低磷酸化的野生型蛋白形成的八聚体结构。 VvWzc_KD在不含Y-cluster时，三种底物（ADP、ADP/Mg²⁺和ADP/Mg²⁺/PO₄^3-）结合状态的蛋白均呈现出经典的“helix-sheet-helix”单体结构，模拟蛋白高磷酸化后八聚体解聚的非工作状态。单体间结构比较发现，ADP/Mg²⁺和ADP/Mg²⁺/PO₄^3-复合物结构类似，但ADP复合物蛋白催化关键残基Lys545、Tyr574发生明显的构象偏转，说明Mg²⁺对活性位点构象变化起到了关键作用。将蛋白单体与八聚体结构进行比较发现，单体ADP复合物与八聚体结构在蛋白催化关键残基和形成八聚体界面的关键残基上均存在明显结构差异。而单体ADP/Mg²⁺和ADP/Mg²⁺/PO₄^3-复合物结构与八聚体催化关键残基结构类似但八聚体界面残基仍然存在较大差异，表明Mg²⁺促进蛋白的催化残基构象由单体非工作状态向八聚体工作状态转变，但八聚体界面关键残基未显示出这种状态转变趋势，推测蛋白的Y-cluster可能对界面的正确形成发挥重要作用。

  综上所述，本研究对VvWzb 和VvWzc_KD两个蛋白进行了系统性的结构和酶学分析。VvWzb以其W-loop独特的结构和功能特点更新了LMWPTP家族现有分类结果，VvWzb代表的新型LMWPTP显示出与真核生物LMWPTP明显的差异，为设计特异性的抗菌药物提供了关键靶点；VvWzc_KD在不同聚合形式下呈现出明显的结构差异，为解决“BY-kinase家族蛋白如何行使功能”这一重要科学问题提供了新的结构线索，VvWzc_KD在真核生物中没有同源蛋白，是开发特异性抗菌药物的优质靶点。

Vibrio vulnificus is an important marine pathogenic microorganism with a wide range of hosts and strong pathogenicity, which can infect humans and a variety of marine economic fish through ingestion and wound contact. The rapid onset and high fatality rate after infection pose a great threat to the development of the aquaculture industry and public health. Therefore, it is urgent to understand the pathogenesis of Vibrio vulnificus and develop specific antibacterial drugs.

The capsule is a special structure on the surface of Vibrio vulnificus, and it is also an important virulence factor that causes the strong pathogenicity of Vibrio vulnificus. However, the structure and function of the synthesis-related proteins have been relatively poorly studied. Vibrio vulnificus Wzb and Wzc (VvWzb, VvWzc) are a tyrosine phosphatase/kinase pair. VvWzc can be hyperphosphorylated by autophosphorylating its tyrosine-rich tail (Y-cluster) at its C-terminus, and VvWzb can dephosphorylate VvWzc to make it hypophosphorylated. VvWzc regulates the production of capsular polysaccharides in the cycle of high and low phosphorylation states, but its mechanism is not clear. In-depth structural and functional studies of VvWzb and VvWzc will provide important clues for the synthesis of Vibrio vulnificus capsular polysaccharides, enhance the understanding of the pathogenicity of Vibrio vulnificus, and provide effective target information for the development of specific antibacterial drugs.

VvWzb belongs to the low molecular weight protein tyrosine phosphatase (LMWPTP) family and contains the conserved catalytic characteristic sequences (P-loop, D-loop), but there is an additional residues sequence (⁴⁰EKSR⁴³) insertion on the key region W-loop relevant for substrate recognition. Further structural analysis revealed that the structure of this region underwent obvious conformational changes after substrate binding, reshaping the shape and surface charge distribution of the active pockets. This is the first time to observe such a significant conformational change of the W-loop in the LMWPTP family. Enzyme kinetic analysis showed that the ⁴⁰EKSR⁴³ insertion region was directly related to VvWzb protein activity. However, its effect on activity is not caused by sequence specificity, and it was speculated that the catalytic ability was improved by the overall spatial effect. Escherichia coli Wzb (EcWzb) W-loop lacks ⁴⁰EKSR⁴³ sequence compared to VvWzb W-loop. Inserting ⁴⁰EKSR⁴³ sequence into the corresponding position of EcWzb W-loop showed that the catalytic activity of EcWzb increased significantly, indicating that the VvWzb W-loop ⁴⁰EKSR⁴³ sequence could be used as a universal "enzyme activity enhancement element" to modify other proteins in this family. Further phylogenetic analysis showed that the LMWPTP family was divided into three types labeled by W-loop type. VvWzb occupies an independent taxonomic position in the LMWPTP family with a unique W-loop type, representing a new type of the LMWPTP family.

VvWzc belongs to the bacterial tyrosine kinase (BY-kinase) family, which contains the conserved periplasmic, transmembrane, and kinase domains of this family. The kinase domain (VvWzc_KD) is an important region for the protein to achieve function through phosphorylation state changes, so this project focuses on this domain. VvWzc_KD presents a C₈-symmetrical cyclic octameric structure when containing Y-cluster, and each monomer binds to an ADP molecule, which mimics the working state of the protein as a kinase. Its Y-cluster exhibits a partially phosphorylated state and is inserted into the active center of the protein, which is very similar to that of E. coli Wzc_KD^K540M nonphosphorylated octamer. It is visually shown that the partially phosphorylated VvWzc_KD still has the structural basis for forming octamers. This is the first time to observe in the BY-kinase family that low phosphorylated wild-type proteins formed an octamer state. VvWzc_KD without Y-cluster exhibit a classical "helix-sheet-helix" monomer structure in the binding state of the three substrates (ADP, ADP/Mg²⁺ and ADP/Mg²⁺/PO₄^3-), simulating the non-working state of octameric depolymerization after protein hyperphosphorylation. The structure comparison between monomers showed that ADP/Mg²⁺ and ADP/Mg²⁺/PO₄^3-complexes were similar in structure, but the ADP complex catalytic key residues Lys545 and Tyr574 underwent obvious conformational deflection, indicating that Mg²⁺ played a key role in the conformational change of the active site. Comparing the structure of protein monomer and octameric, it was found that there were obvious structural differences between the monomer ADP complex and the octameric structure on the key residues of protein catalysis site and the key residues that formed the octameric interface. However, the monomer ADP/Mg²⁺ and ADP/Mg²⁺/PO₄^3- complex structures are similar to the catalytic key residue structures of the octamer, but there are still large differences in the octameric interface residues. It is shown that Mg²⁺ promotes the transition of the catalytic residue conformation from the monomer non-working state to the octameric working state. However, the key residues of the octameric interface did not show this trend of state transition, and it is speculated that the Y-cluster of the protein may play an important role in the correct formation of the interface.

In summary, the systematically structural and enzymatic analysis of VvWzb and VvWzc_KD proteins was performed in this project. VvWzb updates the existing classification results of the LMWPTP family with its unique structure and functional characteristics of W-loop. The novel LMWPTP represented by VvWzb shows significant differences from eukaryotic LMWPTP, providing a key target for the design of specific antimicrobial drugs. VvWzc_KD showed obvious structural differences under different polymerization forms, which provides a new structural clue to solve the important scientific question of "how to function of BY-kinase family proteins". VvWzc_KD has no homologous proteins in eukaryotes and is a high-quality target for the development of specific antibacterial drugs.

第1章 绪论... 1

1.1 创伤弧菌的研究现状... 1

1.1.1 基本特征... 1

1.1.2 感染与危害... 1

1.1.3 诊断与治疗... 3

1.1.4 毒力因子... 4

1.2 创伤弧菌的荚膜研究... 4

1.2.1 荚膜组分... 5

1.2.2 荚膜致病机制... 5

1.2.3 荚膜合成通路中的关键蛋白... 7

1.3 VvWzb与低分子量酪氨酸磷酸酶... 9

1.3.1 功能... 9

1.3.2 结构与活性调节... 10

1.3.3 靶向抑制剂研究... 12

1.4 VvWzc与细菌酪氨酸激酶... 14

1.4.1 功能... 14

1.4.2 结构与分类... 15

1.4.3 细菌抗毒力治疗的优秀靶点... 17

1.5 研究目的与意义... 18

第2章 实验材料与方法... 21

2.1 实验材料... 21

2.1.1 实验试剂及耗材... 21

2.1.3 实验菌株及质粒... 24

2.2 实验方法... 25

2.2.1 目的基因克隆及定点突变... 25

2.2.2 蛋白表达载体的构建... 28

2.2.3 蛋白表达... 30

2.2.4 蛋白纯化... 31

2.2.5 蛋白晶体制备... 35

2.2.6 蛋白晶体衍射数据收集... 37

2.2.7 数据处理及结构解析修正... 37

2.2.8 结构分析... 38

2.2.9 蛋白酶学分析... 38

2.2.10 系统发育分析... 40

第3章 VvWzb的结构研究... 41

3.1 VvWzb生物信息学分析及蛋白纯化... 41

3.1.1 VvWzb生物信息学分析... 41

3.1.2 VvWzb及其突变体蛋白表达纯化... 43

3.1.3 VvWzb聚合形式的分析... 45

3.1.4 VvWzb活性检测... 46

3.2 VvWzb及其配体复合物结构解析... 47

3.2.1 VvWzb及其配体复合物晶体制备... 47

3.2.2 VvWzb及其配体复合物结构解析... 49

3.2.3 VvWzb及其配体复合物结构质量评价... 51

3.3 VvWzb及其配体复合物结构分析... 53

3.3.1 VvWzb及其配体复合物整体结构... 53

3.3.2 VvWzb及其配体复合物活性口袋分析... 54

3.4 VvWzb及突变体活性分析... 57

3.4.1 分光光度法酶动力学分析... 57

3.4.2 免疫印迹法蛋白活性半定量分析... 58

3.5 VvWzb及其同源蛋白比较分析... 59

3.5.1 VvWzb及其同源蛋白结构比较... 59

3.5.2 VvWzb及其同源蛋白功能比较... 61

3.6 小结... 62

第4章 VvWzc的结构研究... 65

4.1 VvWzc生物信息学分析及蛋白纯化... 65

4.1.1 VvWzc生物信息学分析... 65

4.1.2 VvWzc激酶结构域（VvWzc_KD）蛋白表达纯化... 69

4.1.3 VvWzc_KD蛋白聚合形式分析... 71

4.1.4 VvWzc_KD活性检测... 71

4.2 VvWzc_KD-配体复合物结构解析... 72

4.2.1 VvWzc_KD-配体复合物晶体制备... 72

4.2.2 VvWzc_KD-配体复合物结构解析... 74

4.2.3 VvWzc_KD-配体复合物结构质量评价... 77

4.3 VvWzc_KD蛋白配体复合物结构分析... 78

4.3.1 VvWzc_KD八聚体形式的结构分析... 78

4.3.2 VvWzc_KD单体形式的结构分析... 83

4.3.3 VvWzc_KD单体形式与八聚体形式比较... 85

4.4 小结... 87

第5章 讨论... 89

5.1 VvWzb显示出LMWPTP前所未有的结构和功能特点... 89

5.2 VvWzb更新了LMWPTP现有分类... 89

5.3 VvWzc_KD丰富了BY-kinase家族蛋白的结构状态... 91

5.4 VvWzb、VvWzc增进了对于原核生物酪氨酸磷酸化的理解... 92

5.5 VvWzb、VvWzc为创伤弧菌抗毒力治疗提供了药物靶标... 93

第6章 结论与展望... 95

参考文献... 97

致  谢... 111

作者简历及攻读学位期间发表的学术论文与其他相关学术成果... 113