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CwlC是含有同源催化结构域的CwlB（LytC）家族中的细胞壁裂解性N-乙酰胞壁酰-1-丙氨酸酰胺酶。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-末端催化结构域和C-末端区域中的串联重复（重复-1：184-219和重复-2：220-254）组成。生化分析表明，C-末端串联重复序列，命名为CwlCr，可以结合到B。我们试图确定CwlCr的结构以理解肽聚糖结合机制。使用标准的多维异核NMR方法，我们完成了主链和侧链的共振归属，并收集了距离约束和二面角约束。此外，从HNCO（1，2-二氧杂环己烷）实验中得到了明确的26个氢键约束<h3><NC>。使用CYANA进行结构计算，到目前为止获得了低分辨率结构。有趣的是，每个重复采用β α β结构，在重复1和重复2之间形成β折叠。因此，CwlCr折叠可能需要重复序列-1和重复序列-2。结构计算和突变分析的改进过程正在进行中。我们将详细讨论CwlCr与肽聚糖的相互作用的基础上的NMR滴定实验。

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analysis has shown that the C-terminal tandem repeat, named as CwlCr, can bind to the B. subtilis peptidoglycan (unpublished).We tried to determine the structure of CwlCr for understanding a peptidoglycan binding mechanism.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.