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

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analysis has shown that the C-terminal tandem repeat, named as CwlCr, can bind to the B. subtilis peptidoglycan (unpublished).We tried to determine the structure of CwlCr for understanding a peptidoglycan binding mechanism.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