# Crystal Structure of Human Lysozyme Complexed with \(N\)-Acetyl-\(\alpha\)-D-glucosamine
## 3 Results and Discussion
### Structural Comparison of hLZM with Other Lysozyme Structures  
To better understand the saccharide recognition of hLZM, the crystal structure of hLZM-NAG was compared with that of a previously reported crystal structure of hLZM complexed with a tetrasaccharide fragment of the O-chain of lipopolysaccharide-saccharide from _Klebsiella pneumoniae_ (PDB ID: 5 LSH). In this crystal structure, tetrasaccharide bound to the substrate-binding cleft of hLZM with two different conformations (Figure 4a), in which tetrasaccharide was located at the subsites A-D of hLZM. The superimposition of \(\alpha\)-D-NAG-bound and tetrasaccharide-bound hLZM showed a high degree of structural similarity with RMSD of 0.216 A, and two O6 atoms of tetrasaccharide demonstrated positional similarity with that of the \(N\)-acetyl group of \(\alpha\)-D-NAG (Figure 4a). Meanwhile, the ring positions of \(\alpha\)-D-NAG and tetrasaccharide were different due to differences in chemical structure (Figure 4a). Superimposition of these hLZM structures showed that most of the amino acids interacting with saccharide molecules underwent large conformational changes of their side chains on the active-site cleft of hLZM (Figure 4b). Residues Tyr81 and Val128, in particular, demonstrated large differences in side-chain conformation. Subsequently, the crystal structure of hLZM-NAG was compared with those of hLZM complexed with \(2^{\prime}\),\(3^{\prime}\)-epoxypropyl \(\alpha\)-glycoside derivatives of the disaccharides [NAG-NAG-EPO (PDB ID: 1 REY) or GAL-NAG-EPO (PDB ID: 1 REZ)]. In these complex structures, the NAG molecule is located on subsite C (Figure 4c), which coincides with the substrate binding site model. The second sugar of the two ligands (either NAG or GAL) is located on subsite B, but with a slightly different conformation (Figure 4c). This indicates that the sugar recognition mode of hLZM depends on the number and type of sugar. Superimposition of these structures showed that, in the active-site cleft of hLZM, most amino acids interacting  
Figure 3: **Comparison of native hLZM and \(\alpha\)-D-NAG-bound hLZM. ** (**a**) Superimposition of the main chains of native hLZM (magenta), hLZM-NAG1 (cyan), and hLZM-NAG2 (green). (**b**) B-factor putty representation of native hLZM and \(\alpha\)-D-NAG-bound hLZMs. (**c**) Ligand binding in native hLZM and hLZM-NAG1. (**d**) Superimposition of acetate and \(\alpha\)-D-NAG binding from native hLZM and \(\alpha\)-D-NAG-bound hLZM. with saccharide molecules underwent small conformational changes of their side chains (Figure 4d). To evaluate the degree of conservation in the binding mode of \(\alpha\)-D-NAG among the c-type lysozyme family, the crystal structure of hLZM-NAG was compared with that of the crystal structure of \(\alpha\)-D-NAG-bound chicken egg-white lysozyme (cLZM), which is a well-studied model structure of c-type lysozyme. The antibacterial activity of hLZM is three-fold higher than that of the antibacterial activity of cLZM [1]. The sequence identity between hLZM and cLZM was 59% (Figure 5a). To date, three \(\alpha\)-D-NAG-bound cLZM have been deposited in PDB. One of the crystal structures of cLZM (PDB ID: IJA7) was determined in a powder diffraction study [29], while the other two crystal structure of cLZM (PDB ID: 7 BHM and 7 BHN) were determined by serial crystallography performed at room temperature [30]. In the case of the latter two crystal structures of \(\alpha\)-D-NAG-bound cLZM, the results are highly similar due to only differences in the NAG mixing times being used. Accordingly, only one of these crystal structures of \(\alpha\)-D-NAG-bound cLZM (7 BHN) was compared in this study (the one that underwent the longer NAG-mixing time). Superimposition of the \(\alpha\)-D-NAG-bound hLZM with that of the \(\alpha\)-D-NAG-bound cLZM-IJA7 and cLZM-7 BHN had an RMSD of 1.279 A and 0.475 A, respectively, for the main chain of hLZM. The glucosamine group of \(\alpha\)-D-NAG was positioned toward to the  
Figure 4: **Comparison of \(\alpha\)-D-NAG-bound and tetrasaccharide-bound hLZM.