Solution structure of the peptidoglycan binding domain of Bacillus subtilis cell wall lytic enzyme CwlC: characterization of the sporulation-related repeats by NMR.

Bacillus subtilis CwlC is a cell wall lytic N-acetylmuramoyl-l-alanine amidase that plays an important role in mother-cell lysis during sporulation. The enzyme consists of an N-terminal catalytic domain with C-terminal tandem repeats. The repeats [repeat 1 (residues 184-219) and repeat 2 (residues 220-255)] are termed CwlCr. We report on the solution structure of CwlCr as determined by multidimensional NMR, including the use of 36 (h3)J(NC)'-derived hydrogen bond restraints and 64 residual (1)D(NH) dipolar couplings. Two tandem repeats fold into a pseudo-2-fold symmetric single-domain structure consisting of a betaalphabetabetaalphabeta-fold containing numerous contacts between the repeats. Hydrophobic residues important for structural integrity are conserved between the repeats, and are located symmetrically. We also present NMR analysis of the circularly permuted repeat mutant of CwlCr. Secondary structure content from the chemical shifts and hydrogen bonds derived from (h3)J(NC)' show that the mutant folds into a structure similar to that of the wild type, suggesting that the repeats are exchangeable. This implies that conserved hydrophobic residues are crucial for maintaining the folding of the repeats. While monitoring the chemical shift perturbations following the addition of digested soluble peptidoglycan fragments, we identified two peptidoglycan interaction sites of CwlCr at the edges of the protein symmetrically, and they are located approximately 28 A from each other.