【作者】 杨益虎；

【导师】 周丛照； 陈宇星；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 (I)金黄色葡萄球菌富丝氨酸重复蛋白SraP配体结合区(ligand-binding region, BR)的结构与功能研究金黄色葡萄球菌(Staphylococcus aureus)是一种革兰氏阳性菌,它可以导致多种疾病,比如感染性心内膜炎、骨髓炎、脓毒性关节炎以及败血症等。金葡菌表面的SraP是一种富含丝氨酸高度糖基化的蛋白(serine-rich repeat glycoproteins, SRRPs)。SraP可以通过其配体结合区域(ligand-binding region, BR)粘附血小板进而导致感染性心内膜炎。为了阐释SraP与宿主之间的相互作用机制,我们通过X-射线晶体学的方法解析了配体结合区域SraPBR的结构,其分辨率为2.05A。SraPBR的整体结构呈伸展的杆状,由四个分开的模块组成,它们的结构分别与豆科凝集素(Legume lectin)、免疫球蛋白结合蛋白β-grasp折叠以及钙粘素(Cadherin)相似。基于结构分析,结合生物化学、微生物学和细胞实验,可以发现L-lectin模块能特异性地与N-乙酰神经氨酸(Neu5Ac)结合,进而介导SraPBR黏附到A549细胞表面来侵染宿主细胞。而β-grasp折叠模块不具备免疫球蛋白结合蛋白结合活性。另外,C端的两个串联的钙粘素模块与真核生物的钙粘素相似,但是它们的钙离子结合方式不同。小角X射线衍射和分子模拟实验证明SraPBR C端的三个模块通过钙离子维持整体结构的相对刚性,从而将L-lectin模块支出细菌细胞外发挥粘附功能。另外,通过突变实验以及最近鉴定SraP的底物三糖,我们认为SraPBR通过特异性识别唾液酸化的受体来介导金黄色葡萄球菌粘附以及侵染到宿主表皮细胞中。该研究将为开发针对金黄色葡萄球菌的新型疫苗或者抗生素提供理论指导。(Ⅱ) DacB的结构与酶学研究肺炎链球菌中的细胞壁肽聚糖L,D羧肽酶DacB是特异水解肽聚糖肽段L-赖氨酸和D-丙氨酸之间的肽键的酶。敲除DacB基因后会导致肺炎链球菌细胞形态以及隔板分裂的缺陷,暗示DacB在肽聚糖的重塑中发挥重要的作用。因此,DacB有可能作为潜在的抗生素靶标。通过X-射线晶体学的方法解析了DacB的结构,分辨率为2.1A。这是第一个革兰氏阳性菌中的L,D-羧肽酶的三维结构。DacB整体呈球状,由一些个α螺旋及其围绕的核心β-sheet组成。结构分析显示DacB是M15B蛋白酶家族的成员。与M15B蛋白酶家族不同的是,DacB的活性位点有一个六配位的锌离子组成,锌离子与His-Asp-His-Glu四联体以及另外两个水分子配位。通过生化实验发现肽聚糖的四肽(L-Ala-D-iGln-L-Lys-D-Ala)是DacB的合适底物。通过分子对接以及突变实验,鉴定了结合四肽底物的关键残基。基于这些研究成果将为开发针对肺炎练球菌的新型疫苗或者抗生素提供理论基础。更多还原

【Abstract】 (Ⅰ) Structure and function of ligand binding region of SraPStaphylococcus aureus, a Gram-positive bacterium causes a number of devastating human diseases, such as infective endocarditis, osteomyelitis, septic arthritis and sepsis. S. aureus SraP, a surface-exposed serine-rich repeat glycoprotein (SRRP), is required for the pathogenesis of human infective endocarditis via its ligand-binding region (BR) adhering to human platelet. It remains unclear how SraP interacts with human host. Here we report the2.05A crystal structure of the BR of SraP, revealing an extended rod-like architecture of four discrete modules. The N-terminal legume lectin-like module specifically binds to N-acetylneuraminic acid. The second module adopts a β-grasp fold similar to Ig-binding proteins, whereas the last two tandem repetitive modules resemble eukaryotic cadherins but differing in calcium coordination pattern. Small-angle X-ray scattering and molecular dynamic simulation indicated the three C-terminal modules function as a relatively rigid stem to extend the N-terminal lectin module outward. Structure-guided mutagenesis analyses, in addition to a recently identified trisaccharide ligand of SraP, enabled us to elucidate that SraP specifically binding to sialylated receptors promotes S. aureus adhesion to and invasion into host epithelial cells. Our findings have thus provided novel structural and functional insights into the SraP-mediated staphylococcal infection. (Ⅱ) Structure and function of L,D-carboxypeptidase DacBStreptococcus pneumoniae DacB is an L,D-carboxypeptidase hydrolyzing the peptide bond between L-Lys and D-Ala in pneumococcal peptidoglycan (PG). Deletion of the dacB gene has been shown to result in defects in cell shape and septation implying its important role in peptidoglycan remodeling. Therefore, DacB represents a promising target for the development of new types of antibiotics. However, the structure fold and catalytic mechanism of this enzyme remians unknown. Here, we determined the high-resolution structure of DacB in complex with acetate, representing the first Gram-positive bacterial L,D-carboxypeptidase structure. DacB adopts a globular fold, with several a-helices surrounding a central β-sheet. Structure analysis of DacB reveals a new member of the M15B peptidase family. The active site contains an unexpected six-coordinate zinc ion with a His-Asp-His-Glu tetrad and two metal-bound water molecules. We also identified that the tetrapeptide (Ala-iGln-L-Lys-D-Ala) can be used as a favorite substrate by the enzyme. Molecular docking and digestion assays with the tetrapeptide allowed us to identify key residues in ligand binding and catalysis. Based on these findings,we could assign DacB and other L,D-carboxypeptidases in Gram-positive bacteria as metalloenzymes and proposed the identification of specific inhibitors of this important class of enzymes according to a putative catalytic mechanism.更多还原