中国科学院海洋研究所机构知识库

水产病害和养殖环境恶化是危害水产养殖业健康发展的两大因素。本文通过结构生物学手段研究水产养殖病原菌与有害藻类重要蛋白的分子机制，以期为病害发生机理的研究提供结构基础。主要研究对象为：病原菌迟缓爱德华氏菌（Edwardsiella tarda）毒力蛋白溶菌酶抑制因子（Inhibitor of vertebrate lysozyme，Ivy），以及绿潮藻种浒苔（Ulva prolifera）氮代谢关键酶硝酸盐还原酶（Nitrate reductase，NR）。

针对溶菌酶在宿主先天性免疫中的关键作用，细菌相应进化出溶菌酶抑制因子以抵御溶菌酶的杀伤。目前发现的溶菌酶抑制因子分为四个家族，其中，c型溶菌酶抑制因子Ivy通过保守基序“CKPHDC”形成的环状结构来抑制溶菌酶的活性。革兰氏阴性细菌迟缓爱德华氏菌是一种宿主广泛的重要病原菌，其溶菌酶抑制因子Ivy_Et具有与传统Ivy不同的特性，基序形式为“₈₂CQPHNC₈₇”，并且中心组氨酸对抑制活性无明显影响，而半胱氨酸Cys82对其活性具有关键作用。但目前对Ivy_Et的作用机制在分子水平尚未有相关研究。

本研究中，我们分别解析了Ivy_Et蛋白的无底物形式（Ivy_Et-apo）以及Ivy_Et与鸡卵清蛋白溶菌酶HEWL（Hen egg white lysozyme）复合物的晶体结构。结果表明Ivy_Et-apo结构与已知Ivy家族同源蛋白结构类似，呈现经典的helix-sheet-helix的三明治结构；而在复合物结构中Ivy_Et保守基序形成的环状结构以传统“钥匙-锁”的形式与HEWL活性位点区域相互作用，即Ivy_Et的抑制方式与已知Ivy相似。然而不同的是，Ivy_Et具有两种聚合形式，单体和二聚体，二聚体不结合溶菌酶，无抑制活性。将半胱氨酸突变后（突变体C82S、双突变体C82S/C87A），无活性二聚体的比例明显提高，而单体活性与野生型无明显差异，表明二聚体比例的提高导致了C82S抑制活性的下降。

我们进一步解析了Ivy_Et二聚体及其突变体C82S、C82S/C87A二聚体的晶体结构，并利用二硫键交联实验进行验证。研究发现二聚体是通过结构域交换的形式聚合；单体中发挥抑制作用的环状结构被打开，成为结构域交换的铰链区。铰链区中氨基酸通过氢键、盐键相互作用，导致无法结合溶菌酶，从而丧失抑制活性。同源检索比对发现100多个物种的Ivy蛋白基序中两个半胱氨酸绝对保守，暗示着半胱氨酸的重要作用，即通过形成二硫键将环状区域锁住，从而降低发生结构域交换形成无活性聚合体的可能性。本研究首次报道了Ivy的无活性聚合形式，这种聚合形式通过结构域交换的方式形成，为进一步了解Ivy的生物学功能提供了新思路。

绿潮是指大型绿藻脱离固着基后快速增殖形成的生态灾害，不仅危害生态系统、海洋环境，还会破坏沿海水产养殖业，造成严重的经济损失。绿潮在世界范围的海域均有发生，其主要原因种为石莼属（Ulva）绿藻。其中，在中国黄海海域，绿潮主要由浒苔爆发引起。浒苔的迅速增殖与其高效的氮素利用密不可分，其中硝酸盐还原酶发挥着重要作用，然而目前对石莼属绿藻NR尚未有分子水平的研究。本研究中，我们解析了浒苔硝酸盐还原酶第一个电子传递结构域-细胞色素b₅还原酶结构域（UpCbRNR）的结构并对其进行了酶学分析。结构分析表明UpCbRNR包含由6个反向平行的β折叠组成的N端FAD结合结构域，一个罗斯曼折叠（Rossmann fold）组成的C端NADH结合结构域，以及3个β折叠组成连接区域。FAD辅因子位于FAD结构域和NADH结构域之间的裂缝中。UpCbRNR的总体结构以及蛋白与辅因子的相互作用与其同源蛋白相似。酶活性测定结果表明UpCbRNR的催化效率与高等植物CbRNR相当。UpCbRNR是低等植物中第一个解析的NR结构，进一步的同源蛋白序列和结构比对发现了两个具有明显序列长度差异的区域：一个位于FAD结构域，是UpCbRNR所独有的特征；另一个位于连接区域，可用于区分植物、真菌和动物同源蛋白。我们的研究结果从分子水平上加深了对浒苔氮同化过程的理解。

Aquatic diseases and aquaculture deterioration are two major factors that threaten the healthy development of the aquaculture industry. In this thesis, the molecular mechanisms of important proteins from aquaculture pathogenic bacteria and harmful algae were studied by means of structural biology to provide structural basis for the study of disease mechanisms. The main research objects are: lysozyme inhibitor, an important virulence protein in pathogen Edwardsiella tarda and nitrate reductase, a key enzyme in nitrogen metabolism of green tide causative species Ulva prolifera.

To defense the function of lysozyme in host innate immune system, bacteria in turn have evolved lysozyme inhibitors to resist killing by lysozyme. The currently identified lysozyme inhibitors are divided into four families. Among them, the inhibitor of vertebrate lysozyme (Ivy), a c-type lysozyme inhibitor, exerts the inhibitory activity by a rigid loop consisting of a conserved “CKPHDC” motif. E. tarda is an important Gram-negative bacterial pathogen with a broad host range. Its lysozyme inhibitor, Ivy_Et possesses the essential motif in a variant form of “₈₂CQPHNC₈₇”, and the residue Cys82 is essential for Ivy_Et activity while the central histidine His85 shows no effects, which is quite different from the known Ivy. However, the mechanism of Ivy_Et has not been studied at the molecular level.

In this work, we determined the crystal structure of Ivy_Et with no substrate (Ivy_Et-apo) and its complex with hen egg white lysozyme (HEWL), respectively. The structural analysis revealed that Ivy_Et-apo exhibited a classical helix-sheet-helix sandwich architecture which was similar to its homologs. In the complex, Ivy_Et exerted its activity in a traditional key-lock manner with the conserved motif forming a rigid loop protruding into the active site of HEWL, which is similar to Ivy homologs. However, the difference is that Ivy_Et has two polymerization status, monomer and dimer, and the dimer does not bind lysozyme, thereby no inhibitory activity. After mutation of cysteines (mutant C82S, double mutant C82S/C87A), the proportion of inactive dimers was significantly increased, but there was no significant difference between monomer activity and wild type, indicating that the increase in dimer ratio led to the decrease in inhibitory activity of C82S.

To further reveal the mechanism of inactivation, the crystal structures of the dimeric forms of Ivy_Et, C82S and C82/C87A were determined and the disulfide cross-linking experiment was further conducted. It was found that the dimers were associated in a domain swapping manner. In the domain swapped dimer, the loop was formed by the conserved motif changed conformation, and served as the hinge loop which linked the two subunits. As the residues in the hinge region interact with each other through hydrogen bonds and salt bonds, lysozyme cannot be bound, and thus the inhibitory activity is lost. Homologous alignments found that the two cysteines in Ivy protein motif of over 100 species are absolutely conserved, suggesting an important role of cysteines, namely the formation of disulfide bond to lock the loop region, thereby reducing the possibility of domain swapping and the formation of inactive polymers. This study for the first time reported the inactive oligomeric form of Ivy and the feature of domain swapping in lysozyme inhibitor, providing new insights into the biological functions of Ivy.

Green tide refers to ecological disasters caused by rapid accumulations of unattached green macroalgae, it not only harms the ecosystem and the marine environment, but also destroys the coastal aquaculture industry, causing serious economic losses. Green tides occur in many coastal regions and Ulva are a major cause. In the Yellow Sea area of China, green tide is mainly caused by the outbreak of U. prolifera. The rapid growth of U. prolifera is closely related to their high nitrogen utilization capacity, which requires nitrate reductase (NR) activity; however, molecular characterization of Ulva NR remains lacking. Herein we determined the crystal structure and performed an enzymatic analysis of the cytochrome b₅ reductase domain of U. prolifera NR (UpCbRNR). The structural analysis revealed an N-terminal FAD-binding domain primarily consisting of six antiparallel β strands, a C-terminal NADH-binding domain forming a Rossmann fold, and a three β-stranded linker region connecting these two domains. The FAD cofactor was located in the cleft between the two domains and interacted primarily with the FAD-binding domain. UpCbRNR shares similarities in overall structure and cofactor interactions with homologs, and its catalytic ability is comparable to that of higher plant CbRNRs. UpCbRNR is the first determined NR structure in lower plants. Structure and sequence comparisons of homologs revealed two regions of sequence length variation potentially useful for phylogenetic analysis: one in the FAD-binding domain, specific to U. prolifera, and another in the linker region that may be used to differentiate between plant, fungi, and animal homologs. In summary, our data will facilitate molecular-level understanding of nitrate assimilation in Ulva.