# Crystal Structure of Human Lysozyme Complexed with \(N\)-Acetyl-\(\alpha\)-D-glucosamine
## 3 Results and Discussion
### Comparison of Native hLZM and NAG-Bound hLZM  
To investigate how hLZM recognizes the \(\alpha\)-D-NAG molecule, the structures of native hLZM and hLZM-NAG were compared. Superimposition of their crystal structures showed a high level of structural similarity with RMSD of 0.093-0.128 A, indicating there was no large conformational changes upon \(\alpha\)-D-NAG binding to hLZM; however, there was a small shift of the main-chain of the \(\beta\)1-strand and C-terminal helix (Figure 3a). The B-factor representation revealed that the \(\beta\)1-strand, \(\alpha\)5-helix, and C-terminal \(\alpha\)7-helix of \(\alpha\)-D-NAG-bound hLZMs were more flexible compared with that of native hLZM (Figure 3b). Structural analysis of ligand binding revealed conformation changes of Asn62, Arg80, Tyr81, Val128, and Arg131 of hLZM upon \(\alpha\)-D-NAG binding (Figure 3c). In particular, the two methyl groups of the side chain of Val128 of NAG-bound hLZMs were directed more toward the \(\alpha\)-D-NAG molecule compared with that in native hLZM. The side chain of Val110 was separated from the ring center of glucosamine of \(\alpha\)-D-NAG by 0.2 A, indicating that \(\alpha\)-D-NAG-binding induced conformational changes of the Val128 side chain. The side chain of Tyr81 in native hLZM was positioned toward subsite E, but the side chain of Tyr81 of \(\alpha\)-D-NAG-bound hLZM was positioned toward the \(\alpha\)-D-NAG molecule at subsite D, due to rotation of the side change by approximately 106\({}^{\circ}\) (Figure 3d). The Tyr81 residue of \(\alpha\)-D-NAG-bound hLZM interacted with \(\alpha\)-D-NAG via a water bridge (Figure 2c). The smallest distance between the glucosamine of \(\alpha\)-D-NAG and phenol ring of Tyr81 was approximately 5 A, whereas the distance between the ring center of glucosamine of \(\alpha\)-D-NAG and the phenol ring of Tyr81 was approximately 7 A in parallel dispatch. On the other hand, the CZ atoms of Arg80 and Arg131 were separated from \(\alpha\)-D-NAG by 6.40 A and 6.65 A, respectively, resulting in no direct interaction between these atoms and \(\alpha\)-D-NAG. It was considered that the sidechain shifts of Arg80 and Arg131 may have been induced  
\begin{table}
\begin{tabular}{c c c c c c} \hline \multicolumn{6}{c}{**Hydrorogen Bonds**} \\ \hline
**hLZM** & **Distance (Å)** & **Distance (Å)** & **Donor** & \(\alpha\)**-D-NAG** & \\
**Residue [Atom]** & **H-A 1** & **D-A 2** & **Angle (\({}^{\circ}\))** & **Atom** & \\ \hline Ala126 [O] & 3.24 & 3.94 & 130.39 & O1 & \\ \hline Ala126 [O] & 3.60 & 3.92 & 101.36 & N2 & \\ \hline Val128 [N] & 3.79 & 4.08 & 100.14 & O1 & \\ \hline \multicolumn{6}{c}{**Water Bridges**} \\ \hline
**hLZM** & **Distance (Å)** & **Distance (Å)** & **Donor** & **Water** & \(\alpha\)**-D-NAG** \\
**Residue [Atom]** & **A-W 3** & **D-W 4** & **Angle (\({}^{\circ}\))** & **Angle (\({}^{\circ}\))** & **Atom** \\ \hline Tyr81 [OH] & 4.00 & 3.48 & 127.96 & 112.25 & O4 \\ \hline Trp82 [NE1] & 3.02 & 2.72 & 144.92 & 87.42 & O3 \\ \hline Ala126 [O] & 2.79 & 3.71 & 102.23 & 107.74 & O3 \\ \hline Ala126 [O] & 4.02 & 2.72 & 144.92 & 114.21 & O3 \\ \hline Ala129 [N] & 3.01 & 3.23 & 158.53 & 77.42 & O1 \\ \hline \end{tabular}  
* Distance between hydrogen atoms and acceptor atoms. 2 Distance between donor and acceptor atoms. 3 Distance between acceptor and water atoms. 4 Distance between donor and water atoms. 3 Distance between donor and water atoms.  
\end{table}
Table 2: _Cont._by conformational changes of neighboring residues Tyr81 and Val128. Meanwhile, no significant conformational changes of active-site residues Glu53 or Asp71 were observed. Taken together, this suggests that amino acids present in the active-site cleft undergo conformational changes and stabilize \(\alpha\)-D-NAG when \(\alpha\)-D-NAG binds to hLZM.