# Crystal Structure of Human Lysozyme Complexed with \(N\)-Acetyl-\(\alpha\)-D-glucosamine
## 4 Conclusions  
The hydrolases hLZM plays a significant role in the antibacterial and antiviral immune responses in human infants. In this study, the high-resolution crystal structure of hLZM complexed with \(N\)-acetyl-\(\alpha\)-D-glucosamine, a component of peptidoglycan, was reported for the first time. Six subsites for sugar binding were identified in the cleft of the active site of hLZM, with the \(\alpha\)-D-NAG molecule being only observed in the middle of subsites C and D. This result indicates that a single \(\alpha\)-D-NAG molecule prefers to bind near the active site of hLZM. Although the binding mode of \(\alpha\)-D-NAG to hLZM is not fully consistent with that of other hLZM-tetrasaccharide or cLZM-\(\alpha\)-D-NAG binding modes, the position of saccharide binding was similar. This indicates that the specific NAG binding to hLZM and cLZM are distinct, but the subsites C and D are positionally important for the recognition of a single NAG molecule. Meanwhile, the previous peptidoglycan binding and cleavage model of hLZM suggested that the NAG molecule may prefers to interact with subsites A, C, or F; however, the current actual experimental results located \(\alpha\)-D-NAG only in the middle of subsites C and D. This indicates that the modes of binding of a single \(\alpha\)-D-NAG molecule compared with that of the actual substrate molecule consisting of NAG-NAM may differ. Accordingly, further structural and biochemical experiments will be required to identify the interactions between hLZM and their substrates. Nevertheless, this study provided a greater understanding of how hLZM recognizes single \(\alpha\)-D-NAG molecules, which is a  
Figure 5: **Comparison of hLZM and cLZM.****(a)** Sequence alignment of hLZM (Uniprot: P61626) and cLZM (P00698). (**b**) Superimposition of the crystal structures of \(\alpha\)-D-NAG-bound hLZM with cLZM (PDB ID: 1 JA7), which was determined through a powder diffraction study. (**c**) Superimposition of the \(\alpha\)-D-NAG-binding sites of hLZM and cLZM (PDB ID: 7 BHN), which was determined through serial femtosecond crystallography.  
component of peptidoglycan, and how it differentiates it from other sugar-binding LZM structures. The results from this study will provide new insights into sugar recognition of hLZM and the role hLZM plays in the immune system.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12094363/s1](https://www.mdpi.com/article/10.3390/app12094363/s1)]([https://www.mdpi.com/article/10.3390/app12094363/s1](https://www.mdpi.com/article/10.3390/app12094363/s1)), Figure S1: Electron-density map of hLZM after adding NAG and refinement, Figure S2: Electron-density map of hLZM-NAG1 and hLZM-NAG2, Figure S3: Superimposition of crystal structures of hLZM-NAG1 and hLZM-NAG2, Figure S4: Electron-density map of the acetate ion in native hLZM, Figure S5: Interaction between the acetate ion and hLZM.  
This work was funded by the National Research Foundation of Korea (NRF) (NRF-2017M3A9F6029736 and NRF-2021R1I1A1A01050838) and the Korea Initiative for Fostering University of Research and Innovation (KIURI) Program of the NRF (NRF-2020M3H1A1075314).  
Not applicable.  
The coordinates and structure factors for the protein crystals of this study are available in the Protein Data Bank ([[https://www.rcsb.org/](https://www.rcsb.org/)]([https://www.rcsb.org/](https://www.rcsb.org/))) under entries 7 XF6 (hLZM), 7 XF7 (hLZM-NAG1), and 7 XFS (hLZM-NAG2).  
I thank the staff at the 7A beamline at Pohang Accelerator Laboratory for their assistance with data collection.  
The author declares no conflict of interest.