Solution structure and function of Cw1Cr, a peptidoglycan binding domain of a cellwall lytic amidase Cw1C of Bacillus subtilis

枯草芽孢杆菌细胞壁裂解酰胺酶 Cw1C 的肽聚糖结合域 Cw1Cr 的溶液结构和功能

• 批准号: 14560060
• 负责人: SHIDA Toshio
• 金额: $ 1.54万
• 依托单位: Shinshu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14560060/
• 关键词: Bacillus subtilis; cell wall; peptideglycan; amidase; binding domain; NMR structure; 立体構造; 細胞壁溶解酵素

The Bacillus subtilis CwlC is the cell wall lytic N-acetylmuramoyl-l-alanine amidases in the CwlB (LytC) family that contains a homologous catalytic domain. The enzymes are thought to play an important role in mother-cell lysis in sporulation. The CwlC consists of a N-terminal catalytic domain and a tandem repeat (repeat-1 : 184-219 and repeat-2 : 220-254) in the C-terminal region. Biochemical analysis has shown that the C-terminal tandem repeat, named as CwlCr, can bind to the B. subtilis peptidoglycan (unpublished).We tried to determine the structure of CwlCr for understanding a peptidoglycan binding mechanism. Using standard multi-dimensional hetero nuclear NMR methods, we completed the main-chain and side-chain resonance assignments, and collected distance restraints and dihedral angle restraints. Furthermore, unambiguous 26 hydrogen bond restraints were obtained from HNCO(^<h3>J_<NC>) experiment. Structure calculation was performed using CYANA, and a low resolution structure was obtained so far. Intriguingly, the each repeat adopted β α β structure making a β-sheet between repeat1 and repeat2. Thus, it was likely that both repeat-1 and repeat-2 were required for CwlCr folding. The refinement process of structure calculation and mutation analyses are under way. We will discuss the interaction of CwlCr with peptidoglycan in detail based on an NMR titration experiment.

枯草芽孢杆菌CwlC是CwlB （LytC）家族中含有同源催化结构域的细胞壁裂解n -乙酰muramyl -l-丙氨酸酶。这些酶被认为在孢子形成的母细胞裂解中起重要作用。CwlC由n端催化结构域和c端串联重复序列（repeat-1: 184-219和repeat-2: 220-254）组成。生化分析表明，命名为CwlCr的c端串联重复序列可以与枯草芽孢杆菌肽聚糖结合（未发表）。我们试图确定CwlCr的结构，以了解肽聚糖结合机制。采用标准的多维异核磁共振方法，完成了主链和侧链的共振赋值，并收集了距离约束和二面角约束。此外，从HNCO（^<h3>J_<NC>）实验中得到了明确的26个氢键约束。采用CYANA软件进行结构计算，目前得到的结构分辨率较低。有趣的是，每个重复都采用β α β结构，在repeat1和repeat2之间形成一个β-片。因此，很可能repeat-1和repeat-2都是CwlCr折叠所必需的。结构计算和突变分析的精细化过程正在进行中。我们将在核磁共振滴定实验的基础上详细讨论CwlCr与肽聚糖的相互作用。