# Crystal Structure of Human Lysozyme Complexed with \(N\)-Acetyl-\(\alpha\)-D-glucosamine
## 3 Results and Discussion
### NAG Binding  
To investigate the hLZM recognition of the peptidoglycan component, NAG ligand-soaking experiments were performed with hLZM and two crystal structures of NAG-bound hLZM were determined. In the previous peptidoglycan cleavage model, NAG units of peptidoglycan were located at subsites A, C, and E, whereas NAM units were positioned at subsites B, D, and F (Figure 2a). Crystallographic results of the NAG-soaking experiment revealed the electron-density map corresponding NAG was only observed at the middle of the subsites C and D of hLZM (Figure 2b), with the other subsites being occupied by water molecules. The NAG reagent used in this experiment was a mixture of \(N\)-acetyl-\(\alpha\)-D-glucosamine (\(\alpha\)-D-NAG) and the \(N\)-acetyl-\(\beta\)-D-glucosamine (\(\beta\)-D-NAG). Analysis of the refinement results and electron-density map showed that \(\alpha\)-D-NAG was bound to hLZM (Supplementary Figure S1). This indicated that hLZM preferred the interaction with \(\alpha\)-D-NAG compared with that of \(\beta\)-D-NAG when a single NAG molecule was interacting hLZM. To confirm that the NAG molecule reproducibly binds to hLZM, two diffraction data sets of the hLZM crystal soaked with NAG were collected. In the two crystal structures of \(\alpha\)-D-NAG-bound hLZM, the quality of the electron density for \(\alpha\)-D-NAG and the binding site was almost identical, while the quality of the electron-density map for \(\alpha\)-D-NAG surrounding water molecules was slightly different (Supplementary Figure S2). The superimposition of the two \(\alpha\)-D-NAG -bound structures showed that the position of \(\alpha\)-D-NAG was almost identical with the root-mean-square deviation (RMSD) analysis of 0.141 A (Supplementary Figure S3). This indicated that a single \(\alpha\)-D-NAG molecule was specifically bound in the middle of subsites C and D in hLZM. The binding of \(\alpha\)-D-NAG in the two \(\alpha\)-D-NAG-bound structures was analyzed using the crystal structure of hLZM-NAG1, which was superior to that of hLZM-NAG2 in terms of data quality and electron density clarity. The electron-density map of \(N\)-acetyl and the ring of glucosamine were clearly observed in hLZM-NAG1, whereas the electron-density map of the C6-O6 region of glucosamine was weaker. The \(\alpha\)-D-NAG molecule was stabilized by hydrophobic interactions (Val117, Ala126, and Trp127), hydrogen bonds (Asn64, Asn78, Ala126, and Val128), and water bridges (Tyr81, Trp82, Ala126, and Ala129). Specifically, the \(N\)-acetyl group of \(\alpha\)-D  
Figure 1: **Crystal structure of hLZM. ** (**a**) Cartoon and surface representation of hLZM. (**b**) Electrostatics of the hLZM-NAG surface. Subsites in the active-site cleft of hLZM are indicated by the yellow dotted lines. NAG was located at the main chains between residues Asn78 and Ala128 (Figure 2c). The C8 atom of the \(N\)-acetyl group of \(\alpha\)-D-NAG interacted with the CG1 atom of Val117, CB atom of Ala126, and CD2 atom of Trp127 by hydrophobic interactions at distances of 3.60, 3.97, and 3.70 A, respectively. The O7 and N2 atoms of the \(N\)-acetyl group of \(\alpha\)-D-NAG interacted with the amine group of Asn78 and the carbonyl group of Ala126 at distances of 2.92 A and 3.16 A, respectively. The glucosamine group of \(\alpha\)-D-NAG was located at the side chains between residues Asn78 and Val128 (Figure 2c). The O1 atom of the glucosamine of \(\alpha\)-D-NAG interacted with amino group of Val128 at a distance of 3.27 A. The distance between the side chain of Asn78 and ring center of glucosamine of \(\alpha\)-D-NAG was 4.53 A. In addition, five water bridges between \(\alpha\)-D-NAG and hLZM were observed at distances of 2.79-4.02 A. More detail information regarding the distances between hLZM and \(\alpha\)-D-NAG are listed in Table 2.